Science topic

Phosphorylation - Science topic

Phosphorylation is the introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety.
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I want to detect phosphorylation of a specific protein in live cells so I can determine the order of events, however I haven't found a method that would allow this (i.e. signal when phosphorylated). Asking on the off chance someone has any ideas!
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Phosphorylation and dephosphorylation depending on the protein system you are studying may occur on a very fast time scale, and you may just be observing an average condition at best. I have a recent paper on this subject.
and a more detailed Powerpoint presentation on reseachgate explaining my thinking, and putting it in biological contexted.
Good luck.
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I am trying to check the dephosphorylation of a protein by a phosphatase, for that I need to phosphorylate my protein first. Is there any general kinase that can be used for this purpose?
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Unfortunately there is no "magic bullet, one size fits all" kinase. You could include a recombinant form of the kinase (Protein A) in an in vitro kinase assay, thereby eliminating the presence of the phosphatase (Protein C). Inclusion of general phosphatase inhibitors in this kinase assay would also help.
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Hello everyone! I am looking into the phosphorylation level of certain intracellular proteins among different conditions. The way I have been doing it is running two identical gels (with exactly the same amount of loaded samples/same procedures afterwards, etc.), with one for probing for total protein levels and the other for the phospho-proteins. However, the problem is that the two membranes are not even consistent on the signals of loading controls (housekeeping proteins), and therefore I do not think I can reliably work out the phospho-protein/total protein ratio by this approach.
I am not sure if the above situation is common among others, but I have got advised to work on just one gel/membrane and use stripping buffer between probing for total and phospho-proteins.
Therefore, I was wondering if anybody has good experience with different stripping buffers that can remove most primary and secondary antibodies but ideally leave the sample proteins unaffected? Any advice on commercial or homemade stripping buffers will be appreciated. Thank you very much!
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Hello Jian Wang I can only confirm that with my experiment I got almost 100% removal of Ab with this protocol and these timings. You can extend the time to 1hr if you prefer with perhaps 15min intervals instead of the 10. 0.1M glycine pH2.5 usually is great/effective at dissociating Ab-protein interactions and is widely used in such contexts (including Ab purification etc) I can only guess that it will/should work well for yours. As for the blocking buffer, I have found 0.5% casein to be most ideal and often far better than skim milk and in particular with a stripped (reused) membrane might end up being better.
Hope this helps :)
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I am a young bioinformatics student, want to have clues for my project pipeline. hints and expert answers are welcome. THANKS
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I'd start with inferring the amino acid sequence and then BLAST it against proteins of known function. That way you can leverage what is known about the homologous, known proteins to build your starting hypothesis about your unknown protein. You can then design more targeted experiments to test your hypothesis.
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I am using Phos-tag gels for a while now and generally get good results. This time, however, I tried running a Ser-to-Glu mutated protein, where the Ser is usually phosphorylated. I thought I would just lose the phospho-band, but instead it shifted. Has anyone done something similar and could tell me their results? I thought unphosphorylated amino acids do not react with the Phos-tag.
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I recently had a problem on phos-tag page. I am not sure whether it is a shift or not.
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Hello, community.
I wonder why the ratio between phosphorylated protein and total protein is considered rather than the amount of phosphorylated protein.
For instance, drugs A and B simultaneously increase phosphorylation and total protein expression. Drug A seems like stimulate kinase A activity through phosphorylation.
However, the alteration of protein levels under drug B treatment makes me confused about its effect on the activity of kinase A.
What do you think about Drug B? does it stimulate kinase A or inhibit kinase A?
Some papers asserted drug B inhibits the signaling pathway that kinase A involved by decreasing the ratio.
Thank you in advance
Kind regards,
Jinseok Hwang
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If we accept that a given kinase is active only if it is phosphorylated, than its relative activity is correctly described by the ratio of p-Kinase/Kinase level, which is estimated by densitometry on the WB. In this regard, if the activity level, so the ratio of pK/K, is increased by a drug, than this drug activates the kinase, and if another drug decreases this ratio, than it is inhibiting it (maybe better to say anatgonizing with its activity).
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My protein needs PTM like phosphorylation to acquire active form, but it doesn`t have any activity after on column refolding purification with urea.
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All bacteria introduce PTMs, like phosphorylation, acetylation, glycosylation, etc. However, when the protein is overexpressed, the modification is not always observed because of limited PTMs introduction efficiency. Furthermore, if the protein of your interest is not originally from bacteria, it can be not recognized by, e.g., bacterial kinases. Or, it may even be modified in non-native sites. Thus, relying on bacteria to have properly modified overexpressed recombinant protein is quite risky.
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Hi researchers, I work with different variants of a neuronal kinase. In the past, I have been measuring the activity of the kinase via substrate phosphorylation. More specifically, I have been overexpressing the kinase in HEK293T cells and then mixing the lysate with an activating or basal buffer (containing the substrate) before running the samples on SDS-PAGE and subsequent western blot. I have been detecting substrate phosphorylation and the autophosphorylation of the kinase using phospho-specific antibodies.
I am looking for a more high-throughput version of this assay, preferably one that can be run in a microplate. I definitely want to avoid assays that involve P32 or any type of ADP/ATP detection as a readout of activity. Do any of you have ideas?
I have one idea but I'm not sure that it would work. My substrates are GST-tagged, and we have GSH-coated microplates. I was thinking that I could incubate the plate with the fusion proteins, then incubate with kinase-expressing lysate, and then add in my phospho-specific primary antibody and fluorescent secondary. I'm sure there are some caveats to this approach.. What do you all think? Thanks so much!
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Hello Juliana, Wow what a coincidence, i think you perfectly describe our assay :).
Read more about it at www.pamgene.com.
We have developped a 3D microarray that contains numerous kinase substrates (targets), during incubation of a lysate on a array, the active kinases start phosphorilatiing their targets, and we visualize this with a single generic antibody. We can work with very low protein ammounts, ranging from 0,5 - 5,0 ug, and with many sample sourches, from cultures to tissue.
I can share much more, drop me a message on evbreemen@pamgene.com
Looking forward!
Regards, Eddy
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Hello.
I'm working on check protein-protein interaction.
My hypothesis is protein A (kinase) phosphorylates protein B.
So I tried lot of Co-IP bur failed. So I planned to pull-down assay using tagged-protein A and tag antibody conjugated agarose bead.
In this case, I wondered the addition of ATP is required for Co-IP or pull-down assay or not. Is kinase usually form protein-protein interation structure and don't get off from each other?
And someone can recommend protocol of pull-down assay or reference paper?
I appreciate to read mine and hope you have a nice day!
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Maybe the kinase undergoes a conformational change when ATP is available and only the can bind to its target. ATP then gets hydrolyzed quickly while the phosphate is transferred, and you won't find anything in your co-IP.
What *could* work is using a sulfur analog of ATP, ATP-gamma-S instead. It probably will bind, but not be hydrolyzed.
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I need to check wether my Western Blot is able to detect Ser9 phosphorylation of GSK3-beta. Is there a simple method to induce stable Ser9 phosphorylation of GSK3-beta in cell culture?
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Thanks, I decided to try LiCl first but if it doesn't work I will try insulin!
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I'd like to know the different type of post translational modifications that occur in a mammalian cell ?
Few examples from my side are :
  1. Glycosylation.
  2. Phosphorylation.
  3. Ubiquitination
  4. Epigenetic modifications.
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Besides your examples, I would like to add a few more.
Succinylation, malonylation, Sumoylation, S-nitrosylation, Amidation, Hydroxylation, Palmitoylation, Glutarylation, Crotonylation, Sulfation, Formylation, Myristoylation, Glutathionylation.
Please refer to the article below for more information.
Best.
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Usually, paper with immunofluorescence in brain tissue does not perform the ratio of total versus phosphorylated proteins. I wonder why?
W.B = western blotting.
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If accurate information about the state of protein signaling architecture is to be known, adequate preservation of phosphoproteins is required. The activation state of signaling proteins is not adequately preserved in FFPE tissue. Since the formalin fixation process is slow and requires several hours of fixation, during this fixation time, the living cells are reacting ex vivo to stress and rapidly change the underpinning protein-phosphorylation dependent signaling networks from the in vivo biology to a signaling architecture related to the ex vivo state.
Immunofluorescence (IF) allows one to detect the exact location of a target protein within a tissue sample. However, when compared to Western Blot, immunofluorescence has a major disadvantage namely, the stains are not checked against a molecular weight ladder as in Western Blot, which means that there is no way to prove that the fluorescence shown in IF is conclusively related to a specific protein.
Western Blot is a reliable technique for gathering quantitative data. The advantage of Western Blot is that it is effective at generating signal that is proportional to the amount of protein that exists in the sample.
Also, in Western Blot, to account for the possible errors in sample preparation and loading, normalization of samples to remove inter sample/gel variation, target measurements are normalized to reference gene values, thereby reducing loading bias.
So, generally Western Blot would be the right technique when it comes to performing the ratio of total versus phosphorylated proteins as this technique is quantitative, sensitive and far much accurate (steps such as addition of protease and phosphatase inhibitor cocktail to the lysate, normalization etc. are included) than immunofluorescence.
Best Wishes.
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Hi, I want to predict post-transitional modification for phosphorylation. I found lots of websites like Phosida, PhosphoSite Plus. I am just curious about is there any python code for this phosphorylation prediction. If you have, could you share the GitHub link?
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Shaban Ahmad thank you
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)Hi all,
I tried to inhibit pSRC downstream signalling by using SRC kinase inhibitor PP2. In my experiments I could detect a reduction in protein levels downstream SRC, However, SRC phosphorylation levels were increasing upon inhibition.
Did anybody saw such effect when using PP2? or is there any explanation for this?
Thanks in advance.
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The location of Src in the cells may have been changed by PP2.
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Hi everyone, im working on molecular docking and im interested on introducing a phosphorylation on a specific residue of a 3D protein molecule on PDB format, so that i colud evaluate conformational changes. My question is, is that possible? and if it is, which bioinformatic tool could you recommend me for it?
Thanks
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You can also use the CHARMM GUI web site for this purpose, regards
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or if not then please suggest any feasible and cheap method for the detection of phosphorylation.
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Mass spectrometry (MS) techniques would be another method for the detection of phosphorylation. But there are several inherent difficulties for the analysis of phospho-proteins using MS such as signals from phosphopeptides are generally weaker, and secondly, difficulty in observing the signals from low-abundance phospho-proteins of interest in the high-background of abundant non-phosphorylated proteins.
To overcome these drawbacks, several enrichment strategies for phospho-protein analysis by MS have been developed including immobilized metal affinity chromatography (IMAC), phosphospecific antibody enrichment, chemical-modification-based methods such as beta-elimination of phospho-serine and -threonine, and replacement of the phosphate group with biotinylated moieties.
Also, mass spectrometric techniques such as collision-induced dissociation (CID) and electron transfer dissociation (ETD) provide comprehensive parallel analysis of peptide sequences and post-translational modifications like phosphorylation.
Best.
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Hi!
I am trying to get a western blot result with the phosphorylated form of the protein of interest. I know that the protein is produced in our samples and it is around 70 kDa. However, when I tried the antibody recognize its phosphorylated form, I had a band much lower than where it should be seen. It was around 35 kDa. There were also some slightly-seen bands, including where I expected, but they were like non-specific bands.
What could cause it ? What could be the problem ?
Thanks in advance.
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Hello Dudu Gumus
When you are detecting phosphorylated protein in Western Blot, you should keep in mind the rarity of these modifications, the fragility of the phosphopeptide (phosphor ester) bond and also be aware of the risk of artefactual dephosphorylation by phosphatases.
You could do the following:
1. Whenever you are detecting phosphorylated protein by Western Blot use 5%BSA for blocking for 1-1.5 hr. Do not use non-fat dry milk as it contains the phosphoprotein casein which may react with the antibody resulting in non-specific binding and a high background. BSA gives clearer results as it works better with phospho-antibodies as albumin is a secreted protein and it tend to not be phosphorylated.
2. Also, whenever you are detecting phosphorylated proteins, you need to add phosphatase inhibitors like 10 mM sodium fluoride and 1 mM sodium orthovanadate to the lysis buffer just before carrying out lysis to inactivate endogenous phosphatases and protect protein, in addition to protease inhibitor cocktail.
3. Use TBS-T rather than PBS-T in wash/incubation buffer since phosphate ions may lead to interference.
4. Prepare primary and secondary antibodies in 3% BSA in TBS-T.
5. The choice of phospho antibody is crucial. Some factors need to be considered while using phopho-specific antibodies. Phospho-specific antibodies should be affinity purified. They have to be manufactured with appropriate controls and validated in multiple cell lines and species. They should also demonstrate lot-to-lot consistency.
Best Wishes.
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Any published example would be a great help.
Thanks
Amit
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Thank you @Yasuhiro Nishida for your response. You got my question right.
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Hi, I would like to know the concept of Total and phosphorylated protein measurement in western blot in detail.
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I agree with the Dr. Annemarie Honegger, it is dependent on the type of antibody you are going to use for WB. I would suggest you to run the SDS-PAGE having two sets of loaded sample in parallel. Transfer the gel on activated PVDF membrane and determine the extent of transfer using Ponceou stain. Once you are satisfied with the transfer, cut the PVDF membrane in two pieces. Expose the one piece with antibody developed against the protein of your interest (preferable monoclonal), and other piece with the antibody generated against specific sequence of the protein of your interest (that recognize only phosphorylated sequence on the protein of your interest) separately in parallel.
However, while blocking the PVDF to be used for the analysis of phosphorylaion status of your protein of interest, avoid the use of non-fat dry milk or milk in blocking buffer. Milk can specifically react with certain antibodies or detection reagents. Milk contains casein, a phosphoprotein that can react with phospho-specific antibodies. Milk also contains variable amounts of avidin that can interfere with avidin-biotin detection systems. Both of these interactions will result in high uniform background as shown in your image.
Follow the WB protocol keeping all other steps common for the both pieces of PVDF and develop the signal for total as well as phosphorylated protein of your interest.
Carry out the densitometry analysis using Image J software to have quantitative analysis of WB.
Best
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Specifically, i want to capture the phosphorylation event after treating with EGFR from a cell . I also want to see whether my protein of interest is phosphorylated or not. For that, I have to use specific antibodies ( protein of interest) and phospho antibodies. How can I use both antibodies? Western blot or ELISA ? Which will be good for the experiment?
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I agree with Mr. Malcolm Nobre and Dr. Jordan R. Yaron suggestions of going for WB to determine the protein of your interest in cell lysates.
In addition, I would suggest you to prepare the slides of your experimental cells, fix them and then go for ICC using specific antibody against the protein of your interest using fluorescence microscope. This will give one more additional data and advantage that you can track and analyze the sub-cellular localization of specific phosphoprotein of your interest in a cell. You can then perform Image J analysis to evaluate the level of phosphorylation and compare with the control cells.
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Hi Guys, 
I am trying to apply a phosphorylation patch (THP2) to THR residue in VMD, but every time I am having a weird kind of phosphate conformation (please see the attached image).   Is there any problem in my .pgn file, 
topology top_all27_prot_na.rtf
topology toppar_prot_na_all.str
pdbalias residue HIS HSE
pdbalias atom ILE CD1 CD
pdbalias atom GLY OXT OT1
segment P1 { pdb ash.pdb
}
patch THP2 P1:285
regenerate angles dihedrals
coordpdb ash.pdb P1
guesscoord
writepdb AB.pdb
writepsf AB.psf
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Ashfaq Ahmad The problem can be solved following these correction in :
- Topology file
The main problem is, while you are creating your phosphorylated residue, vmd tries to create an "OT" oxygen at the end of the molecule, following the topology as a normal SER. Meanwhile, a normal SER its been created, a pSER has simultaneously been created (with oxygens named O1P, O2P, and O3P). So the connection between oxygen at phosphate organization corresponds only to the creation of an OT between O2P and O3P. Reading the pdb file after its creation by vmd scripting you must find something like these:
ATOM 7518 N SEP P 553 -38.537 8.718 -0.212 1.00 0.00 P N
ATOM 7519 HN SEP P 553 -39.467 9.069 -0.287 1.00 0.00 P H
ATOM 7520 CA SEP P 553 -38.248 7.843 0.882 1.00 0.00 P C
ATOM 7521 HA SEP P 553 -37.231 7.893 1.241 1.00 0.00 P H
ATOM 7522 CB SEP P 553 -39.092 8.219 2.155 1.00 0.00 P C
ATOM 7523 HB1 SEP P 553 -38.857 9.302 2.234 1.00 0.00 P H
ATOM 7524 HB2 SEP P 553 -40.167 8.031 1.949 1.00 0.00 P H
ATOM 7525 OG SEP P 553 -38.722 7.488 3.280 1.00 0.00 P O
ATOM 7526 P SEP P 553 -37.749 8.007 4.367 1.00 0.00 P P
ATOM 7527 O1P SEP P 553 -36.917 9.154 3.897 1.00 0.00 P O
ATOM 7528 O2P SEP P 553 -37.181 6.847 5.030 1.00 0.00 P O
ATOM 7529 OT SEP P 553 0.000 0.000 0.000 -1.00 0.00 P O
ATOM 7530 O3P SEP P 553 -38.612 8.580 5.463 1.00 0.00 P O
ATOM 7531 H3T SEP P 553 -39.191 9.322 5.270 1.00 0.00 P H
ATOM 7532 C SEP P 553 -38.565 6.413 0.498 1.00 0.00 P C
ATOM 7533 O SEP P 553 -39.765 6.137 0.343 1.00 0.00 P O
In bold appears the false OT atom created, forming eventually the triade as a weird phosphate organization. The problem cant be solved by editing this file, erasing the false OT atom, and renumbering the following atoms to be according to the previous ones.
The only solution I found was correcting the TOPOLOGY file, specifically, the phosphoserine topology changing the name of OT atom at patch part in topology file from OT = O3P, O3P its the normal OT name at phosphoserine, but the bivalence between OT and O3P its the main error that's become in a weird conformation.
Must be seen in the topology file like these :
##################################
# PREVIOUS TOPOLOGY PHOSPHOSERINE #
##################################
ATOM CB CT2 -0.08 !
ATOM HB1 HA2 0.09 !
ATOM HB2 HA2 0.09 !
ATOM OG ON2 -0.62 !maintain NA atom type
ATOM P P 1.50
ATOM O1P ON3 -0.82
ATOM O2P ON3 -0.82
ATOM OT ON4 -0.68
ATOM HT HN4 0.34
###############################
# AFTER TOPOLOGY PHOSPHOSERINE #
###############################
ATOM CB CT2 -0.08 !
ATOM HB1 HA2 0.09 !
ATOM HB2 HA2 0.09 !
ATOM OG ON2 -0.62 !maintain NA atom type
ATOM P P 1.50
ATOM O1P ON3 -0.82
ATOM O2P ON3 -0.82
ATOM O3P ON4 -0.68
ATOM HT HN4 0.34
In this manner, the creationg of PDB and PSF files, follow the idea of creation a phosphoserine without OT oxygen, with a normal phosphate coordination.
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Need to establish the assay development of phosphorylation of a transcription factor that contributes to prostate cancer. I want to develop the assay manually rather than using any Kit.
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Very first, try to find out what is already deposited in databases!
A surprising amount of data has found access in publicly available databases, (NCBI.org) often containing information that has never been formally published from mass spec experiments. Look for the latest protein GenBank (.gb) entry of your sequence, it will contain the known information in the annotation section there you may get a first impression on whats known, which can save a lot of unnecessary work . Then you can go ahead and mutagenize the site and investigate your biology. Also Mass Spec is a fast way to map a site, it may also reveal a lot of additional information on other PTMs of your protein of interest:
  • DOI: 10.1016/S0076-6879(02)51853-X
Good luck! Thomas :-)
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How might I break down a protein into its different terminals, e.g., break it down into only its N, middle, or C terminal, or only its N and middle terminal or middle and C terminal?
And to add to that, how might I phosphorylate a proteins phosphorylation sites without incorporating them into its C terminal?
I apologise if this doesn't make much sense, but this is part of a project I've been working on and my supervisor isn't keen on helping me with these questions, but unfortunately I have no idea where or how to start looking for potential answers to these questions as my supervisor also won't give me any pointers.
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yes methods of breadking down protein is am important aspect and when I was in laboratory I remember we use to perform as it is quite simple I can give it an example like while chewing, your body begins to break down and absorb protein. Your saliva contains two enzymes, amylase and lipase, which break down carbohydrates and fats. Catebolites primarily degrade carbohydrate and lipid feedstocks Hydrochloric acid and enzymes known as proteases break down a protein source once it reaches your stomach.
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I have been trying to stably express a kinase in a CRISPR-Cas9 knock out cell line. I have used both pLenti and pHAGE lentiviral plasmids, with the psPAX2 and pMD2.G packaging plasmids. However, even though I have obtained clones that express the kinase, once I evaluate the phosphorylation of its downstream substrates, and blot for its activating phosphorylation, it seems that the kinase is not active. Additionally, after amplifying the kinase gene by PCR of genomic DNA, the sequencing results show several point mutations.
Do you have any suggestions of viral plasmids that I should use? Which ones have you used to add back a kinase?
Thank you!
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Just a couple of observations/questions:
When you say that you got clones that express the kinase, were these virally transduced cells (helps troubleshoot some problems, like vector integrity and packaging issues)? And second, when you are doing PCR from genomic DNA, are your primers specific for your construct of interest? You should also PCR from non-infected cells to see if you get the same result.
Hard to say more without knowing more details (if you are adding back the same kinase in the knockout or something different and how you are designing your primers). Although cells maybe "knockout", they most likely retain almost all of the genomic sequence, and you will need to make sure that you are only picking up your cDNA and not the endogenous gene (depends on the exonic structure of the endogenous gene and your cDNA). There is also the possibility that your "target" is mutated and can not be phosphorylated...may be worth considering some sort of positive control for your system.
As far as vectors go, they are more or less the same, and pLenti and pHAGE have been used well in the past (different promoters will achieve different levels of expression and there are some cell line issues). With a lenti transduction, you should be able to infect your cells with a decent MOI to get at least 80% with at least 1 integration (and you could go for higher, but this can become cell-type dependent) and do your analyses on the whole population versus single clones, at least initially to get your system working.
Long story short, there can be many different reasons, and a lot depends on the experimental setup and conditions....
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Hi. can everyone help me how can I get the band for phosphorylated JNK. briefly I blocked the membrane with 5% bsa in tbst and incubated the membrane with primary antibody (1:1000) with 1% bsa in tbst and incubated the secondary antibody with 1% bsa in tbst. i managed to get the bands for other phosphorylated protiens but not with p-JNK. i also put the phosphatase and protease inhibitor in cell lysate preparation. I sincerely appreciate your comment. thank you
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thank you so much vikrant mehta. sure. i will try it.
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Greetings all.
As you probably know the "Tryptone" used as component of LB media and alike are the product of digestion of casein. Similarly Cas-Amino acids are the product of acidic hydrolysis of casein.
Given the above, and knowing that casein is highly phosphorylated I am trying to understand if the peptides in Tryptone and the amino acids in "Cas-Amino acids" are also phosphorylated.
Many thanks
Yoram
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Yes, Tryptone peptides are are phosphorylated.
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Do you know how to phosphorylate a protein in vitro?
This is the first time for me to modify a protein in vitro and I don't know how to do it at all.
Does anyone know how to do it?
Or I would like to know how to phosphorylate and secrete it in the cell.
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The γ-32P-ATP radiolabeling method is commonly used in the lab, and there are some enzyme-based methods for recombinant proteins in vitro phosphorylation.
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I am aware that TiO columns are used to enrich phosphorylated peptides after digestion of proteins, but can they be used to purify full phosphorylated proteins from crude cell extracts? Something around 30 kDa
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Whereas It is possible
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We would like to phosphorilate enzymatically a synthetic peptide derived from histone H3. Its sequence is QTARKSTGGK. Is there any commercial kinase kits that could be used for this purpose? I have done some research on the Internet, and I only found kinase assays that measure the activity of the kinase. Is there any kit with an enzyme that could be used for global phosphorylation on peptides, as there are enzymes used for digestion or deimination like PADI enzymes? I know that many kinase exists each of them specified for certain substrates. But maybe there are some commercially available kinase specially used for phosphorylation of histones or histone peptides. If you have any information about that, please let me know :)
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Dear all!
Thank you very much for your answers! We would like to further investigate the peptides with mass spectrometry so the efficiency of the phosphorilation would be proven by mass spectrometry. Thank you for all of your advice, PKC seems to be a good choice then :)
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I am looking for a capture antibody to coat on acrylic surface and its corresponding detection antibody related to alzheimer disease detection. I would prefer to purchase an appropriate captuture antibody and detection antibody for phosphorylated tau (p-tau) ps199 target. May I request for your suggestion in this regard?
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Search Google...
For example: https://www.sinobiological.com/, if their portfolio includes your marker of choice ( I have no commercial interest in that company)
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Hi,
I have tried several times to run WB with my protein lysate from heart tissue. My lysis buffer is 1XRIPA +1X of proteinase inhibitor and phosphatase inhibitor cocktails and the samples are always on ice. My samples buffer + adding reduced reagent as DTT before loading on the SDS 4-20% gel. I tried to detect my phosphorylated protein as phospho_phospholamban (Ser16), I got signals but the problem is I got also strong signal in my control samples (from sham mouse ) without any stimulation like ISO or other reagents. Sometime my control samples showing as strong signal as my stimulated samples therefore it is very hard to say the stimulated samples is actually stimulated or not. So I just wonder that do you have any idea why my control samples gave strong signal (band) even I added phosphatase inhibitor? Is it because low amount I did add ? Thanks for help!!
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Hello,
Phosphatase inhibitors are added to the lysis buffer to prevent dephosphorylation of the phosphorylated proteins. This should not be the cause of the problem.
There are two possibilities which I can suggest.
1. How sure are you that the controls you have used are really good controls.
2. The primary antibody may not be specific for phospho-proteins. You may have to change your primary antibody.
Another point I need to add is whenever you have to detect phosphorylated proteins do not use non-fat dry milk for blocking because casein present in non-fat dry milk is a phospho protein which could cause non-specific binding resulting in background bands. Instead, you can use
3-4% BSA for blocking.
I hope this helps.
Good Luck.
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Please recommend buffer to homogenize lungs if I want to study phosphorylation of NfKb, STAT and IRF protein by Western Blot.
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Hi Chintan K Gandhi,
I always use GST buffer, it works very well for phosphorylation.
GST buffer (10mM MgCl2, 150mM NaCl, 1% NP40, 2% Glycerol, 1mM EDTA, 25mM HEPES pH7.5), with protease inhibitors
[1mM phenylmethylsulfonyl fluoride (PMSF), 10mM NaF, 1mM Na3VO4, and protease inhibitor cocktail (Amresco)].
Good luck
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Hello all,
I would like to investigate whether my molecule activates lipolysis via PKC. The PKC activates the MAPK pathway which phosphorylates the hormone sensitive lipase (HSL) at Ser600. However, I can only ever see in papers antibodies against for example Ser660 or Ser565. However, these are only phosphorylated at the PKA or AMPK pathway.
Has anyone worked on this topic before and can help me?
Thanks a lot
Translated with www.DeepL.com/Translator (free version)
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Dear all, I have a set of genes retrieved from mass spec data, where I have the level of abundance and phosphorylation expressed in Log2FC. I would like to sign up and downregulated genes and also, hippo/hyper phosphorylated genes in my network. But I am having a problem loading this data.
I try building the network using string and opening it with Cytoscape, but I couldn't find a way to insert log2FC values that way.
Then I tried downloading string data. csv, and inserting the values there. The problem is that the set of genes is quite large and the data from string is expressed as interaction modules and not as single genes anymore. So I would have to assign manually, which doesn't make any sense.
How would you guys present this data, as an interaction map flagging up and down regulate genes?
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You can opt for the following steps-
  1. First, make a CSV file of your genes with their fold change values.
  2. After constructing your Cytoscape network, import this file as a node table.
  3. Go to the node coloring option and use continuous mapping to color the nodes according to their node weights, i.e. by their fold change values.
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I have been having an issue with my western blots where the bands appear at a completely different size. I have probed for these proteins in the past with success and the same samples have been probed by another colleague with no issues.
These are all phosphorylated proteins and we use a pre-cast Nupage Bis-Tris gel 4-12% with MES running buffer. The ponceau stain (attached) looks fine too.
HELP!
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Why you didn't loaded a negative signal bands? That it could be due to depleted the ECL solution , so all the substrate has been used up before you could get the blot into the imager.
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Can someone working on JNK/p38 signaling pathways explain a little in detail about the phosphorylation of JNK? The SAPK/JNK antibodies apparently detect 2 bands at 46 and 54kDa. I am not very well versed with these signaling pathways so I need help interpreting some western blot data. When one talks about phosphorylation of JNK does it have to be dual phosphorylation i.e. should one expect both these bands to be detected or could it be either one of them also? If I understand correctly the 2 bands represent JNK1 and JNK2 (isoforms). But do they both need to be phosphorylated for JNK to be active? Is there any reference to say that either of the bands is correlated to say pro survival or pro apoptotic function, just as an example.
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Thank you for sharing.
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Dear researchers,
I am new to Western blot analysis. whenever I am trying to absorb the phosphorylation of ACC (Acetyl-CoA Carboxylase) detecting by Western blot, I always observed a double band of pACC.Even several research papers observed double band but none of them explained the reason. Herewith attached the pictures of my membrane which I observed phosphorylation of ACC. Please help me to identify the reason.
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The reason for the double band may be the degradation of the enzyme Or isoforms. We normally use Phosphatase / Protease inhibitor. Do you use both of them? Please try to reduce the SDS-PAGE gel by 6% and try.
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Ideally I would like to be able to image live and also to be able to make fixed slides. I want to probe for the cellular distribution of the variously phosphorylated phosphatidylinositols.
I am trying to go through various companies no luck so far.
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Hi Neal Edward Beeman . Avanti Polar Lipids has high quality lipid products including PI and fluorescent PI.
TopFluor® Derivatives (avantilipids.com)
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I did western blotting to caculate the amount of Phospho-Stathmin 2 (Ser73) and Phospho-Stathmin 1 (Ser16). But I don't know why sometimes Phospho-Stathmin 2 (Ser73) I could see the band and sometimes I coudn't see the band on different mice. I don't know whether phopho-protein is sometimes not expressed depending on the mice?
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Dear Hung Thi Le,
phosphorylation of proteins is a post-translational modification, which either activate or inhibit the protein (in your case Stathmin 1/2). Phosphorylated proteins are not expressed. Proteins are expressed and the phosphorylated or dephosphorylated in dependence of the cellular status.
What you need to interpret your data is staining of your Western Blot with an antibody that detects the unphosphorylated protein Stathmin 1/2. Then you can analyze how much of your proteins are phosphorylated or not (in your case in a possible dependecy of your mouse line). Of course you should also stain actin to look, if your unphosphorylated proteins vary in their expression. So basically, you stain your Western Blots in this order:
1) Antibodies for phosphorylated Stahtmin 1/2
2) Antibodies for unphosphorylated Stathmin 1/2
3) Actin
If you have any further questions, please ask any time.
All the best and stay healthy,
Marc
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Where can I find forcefields for phosphorylated ASP to use with Gromac?
I am trying to construct membrane protein in E2P state but I can't find force field parameters for phosphorylated ASP
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you can try using the Vienna-posttranslational modification tool for any amino acids. In this, you can also select from three different force fields and get the input files needed to perform MD simulations of the modified proteins using the GROMACS package.
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Hi, recently I am trying to build up a DNA library derived from circulating DNA.
I used two-step strategy:
1) cell free DNA were phosphorylated using T4 Polynucleotide Kinase (first phosphorylation);
2) 5-fold of adaptors (unphosphorylated) were ligated with phosphorylated DNA using T4 DNA Ligase (first ligation);
3) Products from step 2 were purified using magnetic beads to remove the free adaptors;
4) After DNA purification, DNA was again phosphorylated using T4 Polynucleotide Kinase (second phosphorylation);
5) After second phosphorylation, the products were further treated with T4 DNA Ligase for nick ligation. Here, we can obtain a cell free DNA library.
As proof-of-concept experiment, I used 60bp blunt end dsDNA and 20 bp adaptor.After the two-step strategy, I run a PCR using universal primers (targeting adaptors), followed by agrose electrophoresis validation. However, the products were all about 40 bp that may be largely from adaptor self-dimerization.
Can anyone help me please? Thank you so much!
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This makes sense. Adapter dimers will always be a huge side product in this style of library construction. You need to be very stringent at the size selection step to exclude them, especially if your library fragments are small. I'm not sure the beads are giving you the resolution you need to separate the 40bp adapters from the 100bp prepared library.
When I've done similar library projects previously, I've used a high percentage, low-melting point agarose gel to purify fragments with. You can isolate the correctly sized band and then use a beta-agarase/ethanol precipitation to recover the DNA. Try this specific formulation of agarose: https://bioscience.lonza.com/lonza_bs/CA/en/Electrophoresis/p/000000000000182176/NuSieve-GTG-Agarose. You can go up to 4-6% agarose with it and get very fine resolution with it (i.e. you can separate bands that differ by 20bp)
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I want to check which MAPKs (p-p38, p-ERK, p-JNK) decrease when i treat with drug in HaCaT cells. How long should I pre-treat with drugs for checking MAPKs signaling at least?
it depends on people. and I have one more questions.
Do MAPKs take a different time to be affected on phosphorylation?
i would appreciate you if you guys give me good replies.
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Hi Jinju,
I agree with Jack, Pratibha and Nickolaos, especially about conditions you choose for looking at for basal phosphorylation. Also, some MAPKs are sensitive to shear forces and will have increased phosphorylation with too much motion.
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When should i add protease and phosphatase inhibitors?
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Freeze the tissue at -80 degrees after packaging. Add lysis solution with inhibitor when taking it out
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Hello. I used doxycycline to induce the expression of my target membrane protein in HEK 293 cells.
After 2 days of adding doxycycline, I removed media, then added the solution containing PKA activator (forskolin or 8-Br cAMP) to treat the cells. I collected the cells and used the cell lysate to do western blot. When detecting by phosphorylation antibody, the phosphorylation level was not changed in the treatment (added forskolin or 8-Br cAMP) and control (did not add forskolin and 8-Br cAMP). I did like this 8 times, all results are the same, with no changes in phosphorylations.
I have also treated the cells after 3 days of adding doxycycline, then did the same as above. The forskolin or 8-Br cAMP treated cell has a significantly higher phosphorylation level than the control. I did like this 2 times, both results are the same, with increased phosphorylation levels.
My boss and lab member said the doxycycline induction time (2 days or 3 days) would not impact the phosphorylation. But I think that cells don't always respond to the signal as we may want: cell confluence and the age of the cells, for example, will impact signaling pathways. I couldn't find a reference for this. Is there anyone who can help me? Any opinions are appreciated. Thanks!
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Hi Zixun
I think that you may perform the same experiment with a longer induction time...if phopshorylation signal increases - the results will be unequivocally against the predictions...Maybe the cells takes some time to respond during induction experiment so you can do the evaluation you did after 5 days of treatment..and see what will happens.
Best regards
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Among the plethora of biological modifications of proteins and nucleic acids, why is the phosphorylation process so universally abundant?
That is, compared to a variety of other possible molecular groups, is there a specific reason for biological systems to adopt phosphorus groups to the extent they do?
Is this just because of the abundance and metabolic economy of this group, or are there other considerations involved (e.g., bond energetics, physical properties, solubility etc.)
Or is this question itself misplaced? Please clarify!
Thank you :)
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Phosphorylation is not the only frequent post-translational modification found in proteins: acetylation is also fairly common. Since such modifications require a high-energy substrate to transfer the modifying group, and nucleoside triphosphates or acetyl-CoA being such common substrates, it is not surprising that phosphorylation and acetylation should be frequently found among modified proteins
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In my studies I want to evaluate heterodimer formation of different STAT molecules after stimulation with different cytokines. What buffer can I use since those proteins are phosphorylated and the dimer interaction (non-covalent) needs to remain intact for the Co-IP.
Can you provide recipes for buffers that you have used for similar purposes? Thank you!
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Hello Nils Ott
You need to prepare the lysis buffer such that it will minimize protein denaturation and at the same time does not affect the target and the associated protein. Please refer to the link below for more information on how to go about with the lysis buffer preparation for Co-IP. You need to add protease inhibitors as well as phosphatase inhibitors in the buffer.
Best Wishes.
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I am looking for the residues of a protein that normally become masked to exclude them as possible phosphorylation sites. There aren't any models yet built for this protein so I was wondering if a tool could predict the residues for me. Thanks
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I am not aware of any tools to predict masked residues and a bit skeptical that such models would give reliable prediction in case of phosphorylations, given that latter can induce protein refolding. May be you can start from other direction and check for identified phosphorylations in databases, like phosphosite.org. Also there are many kinase site prediction tools which may help to identify at which residues phosphorylations may occur (for example GPS 5.0, PhosphoPredict or PhosphoNET.
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Hello, I will be analyzing the active status in the Hippo pathway, and I want to determine the phosphorylation status of the core kinases MST and LATS. My question is: do you think I can interrogate solely for the MST or LATS phospho status instead of looking for the phospho status of both of them?
Thanks for your insights.
Jose Cortes.
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Hello Jose,
I think measuring the phosphorylation status of either of the two kinases could indicates the activation of the Hippo pathway
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Which antibody do you recommend to assess Fyn kinase phosphorylation and activation?
I have seen several publications using this one Phospho-Src Family (Tyr416) Antibody #2101 from Cell Signaling but this also recognizes all other members of the Src family.
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Attaching a paper who have used pFYN from Santa Cruz, for microscopy.
Hope this will work.
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I am treating BV2 cells with my compound and control for different time points: 15-30-60 mins and 3-6-12-24 hours. Then I run Western blot to examine the phosphorylation of JNK and I get strong band even with control sample, like my cells are stimulated. Please see the picture below, the samples are control, my compound at 15, 30, 60 mins and 3, 6, 12, 24 hrs, respectively.
Moreover, when I check the cells by microscope after treatment, I see that some cells are detached from the surface.
Is there any problem with my treatment technique or this is a normal situation that BV2 cells are stimulated after treatment?
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Thank you so much for your suggestion.
I also think that it may be related to cell death because JNK pathway can lead to apoptosis. Actually, when I treated the cells for 12 and 24 hours with this compound, it showed activation of JNK.
And about loading control, I used beta-actin and the bands were quite equal.
So maybe somehow the cells are stimulated right after treatment and they recovered about 1 hour later.
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I am studying a serine threonine kinase. We have worked out a kinase assay for this same protein that involves overexpressing it, lysing and immunprecipitating overnight against a fusion tag, feeding ATP in a kinase buffer in the presence of phosphatase inhibitor, and then western blotting with an epitope specific phospho antibody.
My question is about the potential of doing the same protocol and western blot with pan phospho-S/T antibodies. We hypothesize that in addition to the more well studied N-Terminal phospho-Ser that we have been western blotting against with the phospho-epitope based antibody, that the same protein is likely phosphorylating it's C-terminus as part of an observed relocation away from the membrane.
And 5 residues in the C-terminus have been described in the literature as being phosphorylated, but in non-descriptive high throughput studies. Additionally, there are quite a S/T residues that could be phosphorylated at some point by any kinase.
We have the ability to separately express the C-Terminus and the N-Terminus and the C-terminus does in fact leave its tight membrane association when we co-express the N-terminal kinase domain.
Exactly how common is it for serines and threonines to just be randomly phosphorylated? Without doing more intensive protein purifications, might I see a signal through the background in the way I have described?
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Dear Neal,
S and T should are not randomly phosphorylated, it depends if they are accessible to the respective kinases and phosphatases.
pan- anti pSer and anti pThr antibodies might have very low and non homogenous sensitivity, as pSer and pThr are very small and the amino acid context has a lot of influence if they bind well or not. A good example is the highly specific anti pSer473 antibody that is frequently used to detect phosphorylation of PKB/Akt (in contrast to anti phosphotyrosine antibodies like the famous PY20. Phosphotyrosine is quite bulky).
So, if possible, use antibodies which are specific for the sites you are probing. Check out companies like cell signaling. If the antibodies you need are not commercially available, consider having your own peptide antibodies made against synthetic phosphopeptides and also non phosphorylated controls. When you have several S and T residues in close proximity, you might have to test several combinations, though.
If you are able to isolate your protein by e.g. immunoaffinity chromatography or (2D-) electropheresis, and you have access to a proteomics facility, subjecting it to a tryptic digest followed by MS/MS could give you a lot of information.
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I am currently trying to look for a consensus sequence that's been identified to be a site of phosphorylation by a specific kinase. I am currently using ScanProsite to try and identify whether this site of phosphorylation is found in different proteins from diff. species. For example, the a.a. sequence is P-X-S/T-P (with X being any amino acid). I have tried entering this information followed by the uniprot ID for the specific protein, but I do not believe this is the correct method. Any help would be greatly appreciated!
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Hi Danielle,
Try JalView
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For site directed mutagenesis primer design, specifically those designed to make single-nucleotide substitutions, I have seen methods that utilize back-to-back primer pairs that anneal at the 5' ends via phosphorylation and subsequent ligation; as well as methods that use primer pairs with overlapping sequences on their 5' ends. I am curious if there are any significant differences in outcome between these two designs and what, if any, advantages/disadvantages are associated with either design?
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Ahmet Alptekin - Hi, I saw your other post as well. I'm sorry you had this T missing in your mutagenesis experiment. Because the changes looks consistent I would assume is not a sequencing error although it's always good to get an unequivocal reading. -
Using the Q5 from NEB is pretty much the same with my approach of using custom made 5' phosphorylation. In Q5 you use KLD (kinase, ligase, Dpn1). In my case I don't deal with kinases, the primers are ordered, at a larger mass, are more expensive, but usually I request SDS-PAGE purification or the best purification method they have (FPLC once), the amount of primer you receive is much less than what you ordered because the amount will decrease in the purification step but whatever you receive is likely to be correct.
So I do the inverse PCR with a high fidelity polymerase like Pfu, purify, ligate, Dpn1. Transformation. It's usually very robust.
In your case, since you already have the Q5 kit, I would repeat the experiment but reorder the primers with a purification step, it will be more expensive but not as expensive as if asking for a 5'-phosphorylation. It should work because the primers are bound to be correct. But other options are good too. Good luck.
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Hi,
I want to quantify levels of phosphorylated proteins in some certain samples, and given that doing so using western blot might be tricky, I thought using a ELISA kit for phosphorylated proteins would more suitable. However, I found both kits that quantify p-proteins relative to a standard curve or relative to the amount of total protein. Which would be more adequate?
As far as I understood, it is fine to use the standard curve if this one is made with recombinant purified protein, so I can trust the quantitative value. But I am still not sure how the ratio phosphorylated protein/total protein would be more informative. What I want is just to see the levels of phosphorylated protein in my sample vs control to see if the pathway is being activated or suppressed.
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Eventually you will have to put the amount of phosphorylated protein "X" in relation to someting that is of biological interest in your context. This could be the number of cells, or the dry weight of the cells, or the total volume of mitochondria, the total mass of proteins or lipids or nucleic acids, or the total cell surface ... whatever (I gave some examples that might be sensible in some cases but also almost impossible to determine; just to think about!). To my understanding, this is most often the total amount of that protein "X", as the signal for the cell usually is the relative amount of phosphorylation of this protein. This approach may not be suited when your samples have varying cell compositions (different cell types in changing proportions) or when your experiment deals with differentiating of growing cells (in which ceses it is usually difficult to define a sensible denominator).
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Hi!
I am running a western blot with pJNK as my primary antibody. I use Femto for imaging.
Any ideas why my western blots shows up dirty even with 0.5 seconds of exposure?
It would be really helpful if you have any suggestions. My pJNK antibody and the secondary antibody are both Cell signaling technology.
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Hello Layla
You have not mentioned the details of the protocol which you have been following. Nevertheless, you should keep the following points in mind.
1. The lysis buffer which has been used. Does it contain phosphatase inhibitors and protease inhibitors?
2. For blocking use 3-5% BSA instead of non-fat dry milk because milk contains phosphoprotein casein which will show non-specific recognition of phospho motifs leading to high background.
3. Wash the membrane with 0.1% TBST three times for 15 minutes each.
4. For the transfer process I would suggest you do a wet transfer.
5. If need be you can try changing your primary antibody from another company.
Hope this is helpful.
Best Wishes.
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I would like to evaluate the phosphorylation of Akt in human peripheral blood lymphocytes using western blotting or flow cytometry.
I used PMA/ionomycin or CD3/28 Dynabeads (ThermoFisher) as a stimulant, but could not obtain a positive control.
If you know some stimulants that are useful for phosphorylating Akt in human lymphocytes, I am very happy to be told about them.
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You might try lithium chloride (Licl), an activator of Wnt signaling pathway.
I usually used LiCl at a concentration of 2-5mM for 30-60 minutes for stimulation.
Best regards,
Christian
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I don't need optimization, just an inclusion of the phosphate group would be perfect!
I am working on doing molecular dynamics simulations on a specific phosphorylated protein, but I am having some difficulties finding a method to phoshporylate my PDB model. I was wondering if there are any programs available (aside from PyTMs in PyMOL) that can phosphorylate singular residues in a protein? I cannot seem to get PyTMs to work on either my Linux or Windows computer.
Thank you ahead of time, this would be extremely helpful!
EDIT: Additionally if anyone knows if it is possible to edit PDB files manually or use GaussView that would be great!
- Michael
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No personal experience but you might consider Chimera: https://www.cgl.ucsf.edu/chimera/
Not sure how familiar you are with this program, but you might have a look at for example: http://plato.cgl.ucsf.edu/pipermail/chimera-users/2012-January/007068.html
Best regards.
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I have an experiment that supposedly increases the JNK phosphorylation. However, the phosphorylation increased from basal levels at the p54 band, the p46 band on the other hand was on a plateau in a concentration-dependent manner. When the highest concentration (20uM) was co-incubated with the drug, the cells' viability decreased more. Could there be a difference in the function of the p54 and p46 bands?
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@robin dickinson , the cells was collected after 12 hours. The highest concentration (20um) is the ld50 of the drug. I believe that high jnk would induce apoptosis of the cells but at lower concentrations that only induce low jnk will have protective mechanisms. When i added jnk inhibitor + 20um of the drug, it seems that it killed more cells compared to when drug was used alone. Thus, i think that at early stages, jnk has a protective mechanism.
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I am looking into the Rac1 and RhoA antibodies as they are typical markers for cell migration. When staining the cell, which Rac1 and the RhoA is stained? Does it matter if it is Rac1-GTP or Rac1, RhoA or RhoA-GTP?
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GTP-state is an active form of these proteins. You have to see in literature whether active proteins have different cell localization. Also, it depends on your research question, what you gonna see: to compare active form in some condition and so on
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I have to perform a Western blot of some cell lysates and check if protein x is phosphorylated. In order to do so, I have to use an antibody that specifically recognises protein x (sheep anti-protein x) and another one that specifically recognises phosphorylated protein x (rabbit anti-Phosphorylated protein x). Then I have to use two secondary antibodies to visualize each primary antibody separately: goat anti-rabbit fused with Alexa Fluor 790 and goat anti-sheep fused with Alexa Fluor 680. As each secondary antibody has a different fluorescent protein fused, I can visualize both channels at the same time (680 and 790) and obtain a signal for both antibodies. This way, I can see where does the signal of both primary antibodies colocalize.
First experiments have gone wrong, as I have been getting several inespecific bands in the blot. Could anybody give me some advice? I will try to dilute the secondary Ab to try to reduce inespecific binding. I could also try to increase blocking time. Should I try incubating both primary antibodies separately? And, should I do the same with the secondary antibodies?
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Glad to be of help. I can see your main problem now - Milk!
Milk contains the phosphoprotein, caesin, which causes high background and non-specific binding when staining for phosphorylated proteins.
Use 3% BSA in TBST to block and 1% BSA with your antibodies staining solution instead.
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My 3T3L1 adipocytes are not responding to insulin properly in recent experiments.
I am wondering if my insulin stopped working. Is there a quick test for this?
I normally use 100 nM for 20 min and see an increase in phosphorylation of alt, but lately I do not.
Thank you!
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p-Akt is increased after 1~5min of insulin treatment. You should treat insulin an then cells MUST be frozen using liquid nitrogen after 2 or 5 min.
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I ran a western blot using 12% gel and probing for ERK/pERK (cell signaling antibodies). The green is total ERK, the Red is supposed to be phospho-ERK. I see 2 bands below the total ERK that express the red pERK antibody... but this shouldn't be there.. the phosphorylated protein should be around the same size or higher than the non-phosphorylated ERK... anybody else have this problem? What are these bands?
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Hi there,
Probably degradation fragments of pERK bearing the phosphorylation.
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I am trying to run a molecular dynamics simulation for a ligand that contains a "SEP" residue. also, I am preparing my files using Charmm-GUI using charmm36 ff, but it says that it will rename my "SEP" into SER and will phosphorylate it as a workaround.
My question is will my ligand be the same if this happened? or I need to create parameters and topology files?
hope I will find an answer!
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You can try it and check what you will get in the generated files.
SEP is the phosphorylated serine and couldn't be readable in all FF.
The best
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Dear all,
could anyone suggest suitable markers to detect necroptotic cell using fluorescent microscopy?
I saw that proteins typically used for this are RIPK1, RIPK3 and MLKL. But for each there is a phosphorylated version, so I'm not sure which to buy.
Thanks!
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MLKL is phosphorylated by RIP3/RIPK3.
Phospho-MLKL at Ser358 and Thr357 is the most precise protein to confirm necroptosis via RIPK3-mediated activation of MLKL.
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I did an ICP-OES ( Inductively Coupled Plasma Optical Emission Spectrometry ) test of my cellulose suspensions for checking the phosphorus content in the suspensions. Firstly, phosphorylation of 0.5 g of cellulose was done. After phosphorylation, cellulose fibers were soaked in 100 ml of deionized water (fibers concentration: 0.3 wt%) and ultrasonic treatment was conducted. Then, I did an ICP-OES test for checking phosphorus content in the suspensions. However, they gave me the result in mg/L (85 mg/L; mg = mass of phosphorus atoms and L = Litre ). How can I convert this 85 mg/L to mmol/g (mmol = amounts of phosphorus atoms and g = mass of cellulose after phosphorylation)? Thank you.
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85 x 100/0.5= 1700 mg kg-1
1700/31= ...mmol kg-1
Simply multiply the icp ooes concentration by dilution factor, then divide by molecular mass
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Hi everyone, I am using the pyTMs plug in on Pymol to phosphorylate a particular threonine in my protein. I am having difficulty in selecting only one or two residues - it seems I can only phosphorylate all serines/theronines. Does anyone know how to fix this?
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You can try Maestro software to edit (Phosphorylate) particular residue with energy minimization. Energy minimization is required step while converting (mutate) or editing (like Phosphorylation), so Maestro is a very suitable software for it.
Maestro is also Schrodinger's software like Pymol. You can use free for academic.
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I am trying to quantify phosphorylation of the p65 subunit of NF-kB in primary murine CD8+ T-cells. I stimulated the cells with plate-bound anti-CD3 (1ug/ml) and anti-CD28 (2ug/ml) Abs for 5min and tried to detect p65 phosphorylation by flow cytometry. I did not detect a signal although I was able to get pErk signal at 5min. Is 5min too early for NF-kB phosphorylation?
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Probably the experiment you have to perform is to test a time-course of activation, let say 5, 10, 20, 30, 45, 60, 120 minutes to identify the kinetics of the phenomenon in your system.
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The band downshifts on Western-blot, after kinase assay treatment.
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Post-translational modifications does induce mobility shift of proteins and as mentioned by Jake in majority of the cases it results in upward shift although downward shift is also possible. In my experiments the modification usually causes upward shift of the proteins. To confirm whether your proteins are phosphorylated you can use phospho-specific antibodies.
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I am looking to perform western blot on mouse prostate tissue for phospho -myosin light chain. I have read a few protocols that say to use a slurry of trichloroacetic acid combined with liquid nitrogen to snap freeze the tissues. Does this preserve the phosphorylation better? Can I just use liquid nitrogen? - Thanks!!
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Hi Anne Turco , I used standard protocol (Pro-Q diamond phosphoprotein blot stain kit) to detect phosphorilation of myosin light chain and in my instruction there is no steps with trichloroacetic acid. So I just use liquid nitrogen for freezing tissue.
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Would you please guide me to the in silico steps of performing homology modeling ?? I wanna identify the phosphorylation sites of the human Hsp90 alpha, I looked in the database and I got confused. I would highly appreciate it if kindly could provide me with a link or a chapter to explain this in a clear way. Thanks in advance.
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If you are new to homology modelling, my recommendation is to start with the basics of modelling first.
Here is where you can learn homology modelling using MODELLER: https://salilab.org/modeller/tutorial/
Hope this helps. All the best.
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Actually I am trying to study a protein which is phosphorylated by CDK1. So I cannot use RO3306 but I need to arrest cells at late G2 followed by temporally controlled release into the subsequent Mitotic subphases.
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Hello, S Paul
Maybe ganetespib (STA-9090) - a novel and potent HSP90 inhibitor you use to induced G2/M cell cycle arrest.
Ganetespib (STA-9090) a novel and potent HSP90 inhibitor for its activity in gastric cancer cell lines. Ganetespib significantly inhibited the proliferation of AGS and N87 human gastric cancer cell lines and potently induced G2/M cell cycle arrest and apoptosis.
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My aim is to phosphorylate the proteins (produced by rabbit reticulocyte lysate) via a specific kinase and then remove the phosphorylation via lambda phosphatase (LP). The problem is before LP treatment, proteins of interest and kinases are all dissolved in the buffer. I am afraid the remaining kinases will affect the reactions later. So I want to remove the kinase, and of course not by centrifudge. Then I boiled the samples after kinases treatment on 65°C for 20min. After boiling, there are a lot of red pallets deposited in the bottom of the tubes. Is anyone know what are these pallets?
In my in vitro phosphorylation assays, there are ST buffer, ATP and protein lysates in the tubes.
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Dear Yuling, boiling will denature most proteins in your assay, hence the precipitate.
Ideally, you will be able to immobilize your kinase onto beads (magnetic, sepharose, etc.) so you can easily remove it after the assay. If you have the pure kinase available, it might be easily done with carboxy activated beads (NHS/EDC chemistry), otherwise you could immobilize antibodies against the kinase in that way and use the beads to fish for your kinase.
If you are very lucky, poisoning the kinase with vanadate, arsenate or fluoride might work, but there is quite a risk that this will also inhibit the phosphatase later.
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I need to generate a standard for my pTDP43 MSD assay and an option is to phosphorylate myself a recombinant TDP43 protein with Casein Kinase-1δ. However, I haven't found a protocol to do so. I someone can share a protocol with me it will be really appreciated.
Thank you very much.
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Here is a paper that seems to have what you are asking for : https://doi.org/10.1371/journal.pone.0177834. You can substitute the immunopreciptated protein in their protocol for your recombinant one.
I havent worked with Casein Kinase 1δ, but I have done sucessful kinase assays with recombinant CDK like protein kinases. The conditions I use are as follows: 10x kinase reaction buffer (1 ml) : 400 mM Tris-HCl pH 7.5, 100 mM MgCl2, 1 mM EGTA, 10 mM DTT and 1% phosphatase inhibitor (I use Sodium Orthovanadate). Some kinases require 2mM MnCl2. Components of each kinase reaction : 10x reaction buffer 3 μl, Kinase (1ug) 1 μl, Substrate (same molar ratio as kinase) 1 μl, Deionized H2O 22.7 μl, ATP mix 2.3 μl. Total 30 μl.
Good luck.