Phosphorylation - Science topic

HI,

your antibody could be innefective in recognizing your protein by IP; the epitope can be masked by other interacting protein or be accessible only on denatured protein (western blot...), try another antibody, try to put a tag on your protein...

Is the specific phosphorylation observed at tyrosin or a different amino acid is phosphorylated?

Generally speaking, using MS-based omic is sufficient to validate PTM identification...

If protein level validation is needed, the suggestion given above is adequate if the phosphorylation is at tyrosine residue...

If peptide level site-specific PTM must be verified, other than MS-based analysis you may use dot blot or ELISA assays after peptide fractionation and conjugation with the purposed peptide to an anti-p antibody to conclude if a specific antibody is found...

For the protein level validation PTM PTM-specific staining may also be OK after PAGE application but site-specific information is lost indeed...

You may use phosphatase treatment to remove phosphate moieties and next you may observe the mass shifts to prove the PTM and its bound peptide location...

Last but not least; you need to use ETD alternative fragmentation to get c and z ions to validate the phosphorylation site (amino acid level) and this may exclude the need for any antibody-based approaches or any further validation...

Good luck, İEA...

Hi Trevor Price ,

I've tried with Phospho-Tau Family Antibody Sampler Kit #96628 (Cell Sign.) without succes.

Incubation time:

Primary Ab - 12 -16h., overnight

Secondary: 1h, RT, dillution: even the 1:3000 was not efficient.

Wet transfer, overnight, 35V in 4°C.

I prefer blocking with 1% BSA in TBST, not milk.

In our hands, pIC activates pIRF7 not 3 (see PMID: 37212952). We can trigger pIRF3 by LPS stimulation (3 h and 100 ng/ml). Hope this is helpful!.

Hi Fatima,

You might also try running separate, replicate gels, one for phospho- and one for total protein. Instead of stripping and reprobing.

Also, are you 100% confident in the specificity of the phospho antibodies? Is it possible the bands for pSTAT1 and/or pSTAT3 are really aomething else?

I appreciate your response. I do not think so. We are utilizing a PI3K inhibitor. I am currently in the process of investigating to understand the cause of these variations. However, I have been unable to discover any relevant information thus far.

Wolfgang Schechinger Thanks for your answer!

Yes, that is possible.

Thank you very much for the link, it is very interesting. I've now been retired for a few years but still follow areas I once worked in, and even occasionally still publish.

Good luck with your research.

Phil.

Thanks for your informative response.

And to make to clear I used airway epithelial cells in vitro and the stimulation was done by using IL-13. What I also found in literature is that the peak of STAT6 phosphorylation is at 30 minutes of stimulation.

You can use charmm-gui to prepare your system... They have parameters for pretty much all phosphorylated aminoacids

Thank you very much! I really appreciated your help.

Hello

Would recommend this research article :

Kind regards

My lab has done non-radioactive quantitation of phosphorylation of specific substrates by Western blotting. You might want to take a look to see if this approach would work for you.

A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications

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.

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.

Hi Jian, personally I never use stripping in this case, because most of the time stripping is not completely cleaving off the first primary antibodies and you might still get some old signals. On top of that, stripping is also removing to some extent the proteins from the membrane. What I do usually is that I detect phosphorylated and total proteins on two different membranes (parallel blots coming from the same sample preparation). This you can later normalize each blot to a housekeeping gene and do quantification. Alternatively, you can use LICOR (even if your primary antibodies are coming from the same species, you can use LICOR and detect first Phospho form, subsequently detect the total form with a different channel).

Hope this helps and good luck!

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.

I recently had a problem on phos-tag page. I am not sure whether it is a shift or not.

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).

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.

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

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.

Thanks, I decided to try LiCl first but if it doesn't work I will try insulin!

Hello Vikram Kumar

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.

Hello Lucas Kniess Debarba

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.

Shaban Ahmad thank you

You can also use the CHARMM GUI web site for this purpose, regards

Hello Mohd Sarim Siddiqui

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.

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.

Thank you @Yasuhiro Nishida for your response. You got my question right.

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

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.

Best

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 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.

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

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.

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:

Good luck! Thomas :-)

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.

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....

thank you so much vikrant mehta. sure. i will try it.

Yes, Tryptone peptides are are phosphorylated.

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.

Whereas It is possible

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 :)

Search Google...

For example: https://www.sinobiological.com/, if their portfolio includes your marker of choice ( I have no commercial interest in that company)

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.

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

Check https://www.cellsignal.com/products/primary-antibodies/phospho-hsl-ser660-antibody/4126

You can opt for the following steps-

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.

Thank you for sharing.

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.

Hi Neal Edward Beeman . Avanti Polar Lipids has high quality lipid products including PI and fluorescent PI.

TopFluor® Derivatives (avantilipids.com)

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

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.

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)

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.

Freeze the tissue at -80 degrees after packaging. Add lysis solution with inhibitor when taking it out

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

The fully overlapping primers work well in general I use this method all the time because it is simple and does not involve a ligation step. If the PCR product yield is good, after DpnI digestion the yield of mutants is >85%. However, sometimes the primers anneal better to each other than to the template sequence. One way to avoid that is performing 5-6 amplification cycles with one primer alone, then mix the reactions and continue amplification for another 13-18 cycles. This is quite efficient and in most cases will solve the problem. If that doesn't work either, then I go for the non-overlapping primers and ligation.

Cheers !

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

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.

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.

Hello Jose,

I think measuring the phosphorylation status of either of the two kinases could indicates the activation of the Hippo pathway

Attaching a paper who have used pFYN from Santa Cruz, for microscopy.

Hope this will work.

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.

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.

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).

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.

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

Dear Michael Bakker ,

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.