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Hi,
I need to find a server that docks my peptide sequence against the PDB database and find a probable target for me?
Best wishes,
Zahra Kayani
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Thanks a lot
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I want to simulate a histone protein -peptide complex. can i use a dodecahedron box.
Expert opinion is highly encouraged.
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Shefin Basheera Assalam o alaikum wa rahmatullahi wa barkatuhu
dodecahedron water box model is standard and dodecahedron fully occupied histone. If any doubt you can run command for water box and visualize through DS viualizer.
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I am computing Van der Waal interactions in python for a peptide of size 10 residues for various conformations. The total conformations (or the number of PDB files is 300,000). Is it possible to compute only the 1-4 atom distances to compute Van der Waals interactions as the bonded and 1-3 atom distances are irrelevant when it comes to Van der Waal interactions using some python module?
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Hi . I was wondering if anyone could help me determine N terminal and C terminal end of a new peptide sequence that I'm looking to synthesise.
for e.g.
1. if DIEFRVLH is the peptide sequence I'm interested in, how can I determine the ends and directionality of these peptides?
2. What programs/softwares are available to study more characterstics of peptide and to determine its drug potential? I have already used ProtParam and Swiss ADME program.
Thank you
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David Salehi Thank you sir for your response to my question. I want to investigate the cytotoxicity of some paptides designed in silico on mamalian cell lines. So far, all I have are the sequences themselves and the purity. I used ProtParam and SwissADME to determine some physicochemical properties of these peptides such as Molecular weight, instability index.
I was looking for more information on peptides based on their sequences that would help me design the test. Do you have any recommendations on what I should keep in mind or look for while designing the test as it is my first time dealing with peptides?
Thank you for your paper. It helped me understand the concepts more.
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when I docked peptide and protein in the patch dock, it gives me ten best structures. should I work with all these ten structures?
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You can choose several poses that appear by considering the ACE value. You can discuss the quality of the interaction based on the obtained ACE value.
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Is Pro-collagen 3 N-terminal Peptide (P3NP) a fragment of Pro-collagen 3?
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It is somewhat confusion since both Pro-C3 and P3NP (or better PIIINP) are called “N-terminal propeptide of type III collagen” since both are N-terminal extensions of collagen type III.
Have a look at the enclosed picture (for complete chapter see enclosed file).
Hope this clarifies it all: PIIINP is part of Pro-C3.
Best regards.
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I want to perform a molecular dynamics simulation of a peptide inhibitor in complex with a protein from Leishmania donovani (pdb id: 3t4p). The only protocol I have is a protein-ligand complex simulation in GROMACS with a CHARMM force field package. I want to know if this same package can be used to run the peptide-protein dynamics.
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Thank you Bettina, the information has been useful.
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Hi,
I cannot purify a peptide synthesized by solid-phase peptide synthesis using HPLC or Biotage. I tried both ways, and still, the trace is not pure. What can I do differently to get a pure peptide fraction?
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your question is opaque. But all the things depend upon the protein sequence
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Dear all,
I'm running a nanoLC-MS/MS system (from Thermo Fisher), while for all samples (peptides), we only observe high TIC at 95% buffer B (acetonitrile), nothing was eluted at 2-50% buffer B in 60 min. the TIC chromatogram of samples were similar to the blank one (pure water), although we find different MS spectrum. Whet we have done:
(1) Clean the whole system with 70% buffer B, actually NC pump and loading pump pressures kept quite normal.
(2) Sample injection model is micro-pickup, we use buffer A (2% ACE/water) as the transport liquid.
(3) Previously, during elution, the NC pump flow is from valve 3 (in)-2-5-4 (out) then to the analytic column. The trap column was set between 5 and 2 (flow direction 5-2, sample loading was from 6 (in)-5-2-1 (out, discharge liquid). Therefore, in this way, the sample concentrated in the trap column was reversely washed out during the elution step. Actually, I feel very confused about this, can the trap column be operated at a reverse flow direction?? Therefore,
(4) I changed the flow route during the elution, i.e., 4 (in)-5-2-3(out).
(5) None of above step works, so, I thought the trap column might be damaged, and then replace it with a new one, exactly the same P/N.
Then, again, it does not work.
Regarding the samples, we detected the protein concentration, which were around 2 mg/mL, 2 or 4 ul of injection volume. All the samples were checked using HPLC, most peptides were eluted before 30% buffer B.
What I'm thinking:
(1) The analytical column is damaged? while, as I know, if nothing blocked, such a column should work for a long time, right? If yes, what is the reason for the damage of analytical column?
(2) The trap column should work at a reversed flow direction during the elution??? ( I did not try this for the new trap column). If so, what is the reason behind??
Any tips, comments, helps are welcome, MANY THANKS in advance.
Best,
Yuhong
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Hello Yuhong Mao,
Perhaps try a few injections without the use of a trap column. This will identify if the analytical column is functional or not.
Once you have determined the analytical column is functional, add a trap column but install it so that the loading and eluting directions are the same i.e., do not operate in a reverse flush way as many columns no longer support this.
Finally, ensure that the column switching timings are correct so you have adequate time to transfer the sample to the analytical column.
Good luck.
Best,
Peter
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Hi everyone, I am currently in the process of designing a stapled peptide. The sequence itself is very hydrophobic, thats why I am currently measuring an comparing the helicity in TFE/water (1
:1). The unstapled peptide is showing good helicity since the solvent mixture is already inducing/stabilizing the alpha helix. Measuring my stapled peptide now (stapled in i,i+7 position with reported suitable linker) is giving me a lower helicity. Are there cases where stapling a peptide is not increasing the helicity of the peptide compared to the unstapled version? Could this be an effect of the solvent mixture and the hydrophobicity of the peptide?
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Based on your information it is hard to say what is going on here. I think it is too simplistic to say that stapling of a peptide always increase helicity, I think it is fairer to say that it frequently contributes to stabilizing helicity. As indicated here https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832507/#B29-molecules-24-03654 and I quote “Making a comparison between the properties conferred to a peptide by a stapling bridge and another is not trivial. Many factors, as the stapling position, the nature of peptide, or the length of the staple brace contribute in determining the properties of the stapled peptide”
Figure 7 (and the discussion about it) indicates something interesting “The results obtained of the two different research groups are quite consistent, showing the same general trend for both the classes of peptides (Figure 7). In both cases the circular dichroism analysis performed in a lipophilic medium showed that the peptides have more or less the same helical content of the non-stapled one”
I see resemblance with peptide research looking at reasonably hydrophobic peptides (like signal peptides and transmembrane peptides. It is reported that in (pure) water highly hydrophobic peptides show a random coil, see for example SP of prePhoE:
While in the appropriate amphitropic/lipophilic environment it shows significant helicity:
I think (since you stress the hydrophobicity, and the linker might contribute an even higher hydrophobicity) your stapled peptide tends to aggregate. So, I would try:
-Pure water
-SDS solution
-If not a single positively charge is present in your sequence you could try a neutral detergent like DDM or DPC instead of SDS, see also https://www.sciencedirect.com/science/article/pii/S000527361200017X
-The approach indicated by John Carter might be informative as well (I would predict no difference between the two at higher TFE content if there is there might be a serious solubility issue with the stapled version)
Best regards.
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Hi everyone! I want to do a chemical reaction that involves the sidechains of arginine and lysine.
The desired chemical reaction is a molecule with COOH groups reacting with Amino groups of the amino acid sidechains of lysine and arginine.
Unfortunately, Lysine and arginine have the alpha-amino-groups, which normally are forming the peptide bond. Those will also react with the COOH groups of the molecule...
So my plan is
1) block alpha-amino-groups in lysine and arginine
2) now add the other molecule and do the desired chemical reaction
I am a geneticist, not a biochemist. I would highly appreciate ideas how to chemically react the alpha-amino-groups away (block them). Possibly with acetic acid that reacts with the alpha-amino-group?
I was thinking of going to a specific pH near the isoelectric point, where the alpha-amino-group is not protonated. Thus the alpha-amino-group might react with the molecule, while the amino groups in the sidechain do not??
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It would be good to know more about the purpose of your experiments. As already mentioned, arginine is not easily derivatized this way. Lysine can be purchased in alpha-protected form, e.g. CAS 1946-82-3 (non-cleavable), or CAS 105047-45-8 (cleavable).
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I am looking to get a peptide synthesized and have gotten many quotes from various companies. One company, WatsonBio, provided a very appealing quote, so it seems too good to be true. I am trying to do some recon on the company, but I thought I'd reach out to this community to see if anyone has worked with them before.
Thank you in advance!
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Hi Judith, I did not go with them. I went with GenScript and was happy with their product
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Dear all,
Could you please advise me on what would be a typical composition of neopeptone?
I understand that it provides vitamins, nucleotides, minerals, and peptides, but is it possible to get more detailed information?
Thank you very much in advance,
Olga.
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Malcolm Nobre thanks very much for your kind help!
Yes, the table on pp 42-43 looks helpful, many thanks!
It would be great to have a breakdown of carbohydrates though...have you ever come across it?
Thanks,
Olga.
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Im willing to do peptide sequencing of my sample using edmand degradation method for which i need to characterize and purify the peptide sample,I have access only to 2D gel electrophoresis and HPLC ,will there be diffference in the results or purity of the sample using these techniques when comparing with mass spectrometry analysis
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The oldest of old school methods. By separating by 2D-PAGE, you need to get the protein out of the gel, which means either blotting to PVDF or ingel digestion to peptides. Blotting to PVDF means that your Edman will give you the amino acid sequence from the N-terminal which may be enough to give you a match. Ingel digestion means that you have peptides that you then need to separate by reversed phase and then Edman separately which you could then use to reassemble into the protein sequence. However, the won't be a difference in the results obtained if your sequencer can reliably give you 10-20 amino acids. But it will take 50-100x longer than LC-MS/MS. Edman cycles are typically 1 hour per amino acid whereas LC-MS/MS for a single spot is ~30 mins using nanoflow.
Adding to the complexity of this, there are actually many proteoforms in a single 2D separated spot.
Are you able to perform an in gel digest and send the peptides to someone who can do the LC-MS/MS for you? It would save an enormous amount of time and expense compared to the Edman.
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I'm looking for the best/most suitable code of column /resin to use in chromatography steps for separation of freeze-dried peptides in the ÄKTA pure. The manufacturers brochure has about 5 to 6 options, but I could not find what is the best for may case. Someone has experience int this case and this device? The fractions were ultafiltraded in MWCO raging from 10 to 3 kDa. and kept freeze dried in -80 °C
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Can the AKTA Pure tolerate solvents used for reversed phase? Or is there a reason that you don't want your samples to be in Acetonitrile (or Methanol) and either TFA or Formic Acid? Reversed phase is going to give you the highest resolving power for separating peptides.
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I have encountered some problem when doing md for protein-peptide. Gromacs cannot recognized the peptide as the peptide also composed with benzoyl group. Any suggestions for my problem?
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Yes, always use the same (or comparable such as AMBER + GAFF) force-field for all components in your model.
You would basically create a modified AMBER force-field consisting of the standard parameters + the ones for the modified residue, so you should be able to use that force-field for both components.
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Hello everybody!
I'm performing my Master degree in biochemistry and I had to to find a way to map the interaction between 2 proteins (so that it will be then possible to design a specific peptide inhibitor to disrupt this bond), but I do not know how to proceed.
Does somebody know an experimental approach to map a binding site between two proteins precisely ?
Thank you very much in advance for your help!
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The best method is to crystallize the complex and obtain a high-resolution x-ray structure. Nowadays, it is also possible to obtain almost as high resolution of protein complexes without crystallization using cryoelectron microscopy. Both of these are highly specialized techniques, which will probably require forming a collaboration. The first step is to obtain the complex in a highly purified form.
Computational techniques are beginning to be available for modeling the structure of protein complexes using the primary sequences.
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Dear all,
I am looking for a LC-MS spike-in dataset with :
- two or more classes.
- at least a hundred of samples.
- a list of the spiked peptides (mz and RT).
I found a dataset in Leepika Tuli et al, 2012 (https://www.researchgate.net/publication/221865421_Using_a_spike-in_experiment_to_evaluate_analysis_of_LC-MS_data) which corresponds perfectly to what I am looking for, but it contains only 10 samples.
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in my project i want to investigate the proteolytic cleavage of a specific peptid through skin bacteria. For that i firstly want to collect the proteases of the bacteria and measure concetration with the Bradford-Assay before i incubate them with the peptid of interest.
My problem is that i cant really get the bacteria (mainly Staphylococcus) to secrete sufficient amount of the protease.
I tried to incubate the baterial pellet for various duration after suspending it in PBS (pH = 7,6). The amounts which i got from where all pretty low ranging from 0,23 mg/ml - 0,028mg/ml.
Does anyone have any idea how to get larger amounts.
Thank you for your time and help.
Kind regards,
Philipp
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Just a thought: Have you considered growing the bacteria in a medium in which the only source of nitrogen and amino acids is in the form of whole proteins?
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We take a na-caseinat sample and digest it for 15 min with trypsin. On SDS-gel (stained with Coomassie) we can nicely see the fading of the main band.
When using comparable samples for bradford assay, we can also see the digestion of the protein. My question is now, why do we see this, as the content of aminoacids (in the form of large protein or smaller peptides) stays the same and bradford stains does not peptide bonds?! Thanks.
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The development of color in Coomassie dye-based protein assays has been associated with the presence of certain basic amino acids (primarily arginine, lysine and histidine) in the protein. Van der Waals forces and hydrophobic interactions also participate in the binding of the dye by protein. The number of Coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein.
Free amino acids, peptides and low molecular weight proteins present in the digested sample do not produce color with Coomassie dye reagents. In general, the mass of a peptide or protein must be at least 3,000 daltons to be assayed with this reagent. This is the reason why there is variation in the response of the Bradford assay which is the main disadvantage of this method.
So, the Coomassie (Bradford) Protein Assay is used to measure “high molecular weight proteins”.
Best.
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I am trying to complex small peptides (~7000-9000 MW) with plasmids (roughly 6000 bp) to condense the later down to form particles. Recently, I've run into an issue when trying to concentrate my materials. If I use a higher concentration of plasmid and scale the peptide to it (using N/P ratios) a precipitate will form in solution after mixing. The solutions do contain some salt in the form of PBS and the precipitate does lessen when the salt is removed. Does anyone have any tricks for getting these materials to play nicely with each other and stay in solution? Thanks!
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The mixing order could have an effect, compare protocols for the use of transfection reagents. Another option might be to add reagents that complex charged groups and will shield them. Possibly, stuff like (bridged) trialkylamines can do the trick.
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The Dyna-bead (M-280) protocol calls for a PBS with 0.05% Tween-20 solution to be used to wash and elute mAb labeled beads. Ultimately these beads will be used to enrich peptides out of samples for detection by mass spectrometry. Will the Tween contaminate my peptide samples and inhibit ionization? Is it necessary to use the detergent or can a different one be used?
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Thanks you for your reply. I will definitely try it out.
Regards,
Jon
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Does anyone know if oxytocin can be measured in body fluids (specifically saliva) using Fourier-transform infrared spectroscopy?
Thanks
Phuoc-Tan
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:-) You're welcome, Phuoc-Tan Diep ! I’m glad I could help.
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I want to use it in a project, peptibody project, that we use an Fc-region attached to peptides.
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You could try UmabDB (The Antibody Therapies Database). A 7 day free trial is available.
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I want to synthesize a peptide of 13 amino acids. Tell me some company to purchase a peptide.
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From Shilpa Medicare
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Triplicate peptide samples were dissolved in human serum at a concentration of 100 microM and incubated at temperature of 37 C. Then, the aliquots of each peptide were taken out at 0, 12, 24, 36, and 48 h. Each aliquot was quenched with 6 M urea and incubated for 10 min at 4 C. Then, 20% TFA was used to precipitate serum proteins for an extra 10 min at 4 C. All aliquots were centrifuged at 14,000 g for 15 min, and the supernatant was analyzed by RP-UPLC using a linear gradient of 5–30% buffer B for 5 min.
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The peptide hypothetically interacts with serum proteins to undergo a reaction. You add urea to break any residual binding of peptide, so it doesn’t inadvertently come down with protein partners.
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Hello all,
I am attempting to express a Plasmodium gene which is extremely AT rich with several repetitions of the same. The peptide I am attempting to express is 506 amino acids long.It has 2 distinct overlapping domains(which i am trying to express together). However I have tried Ecoli BL21(24*C for 12hrs) and Arctic strains(12*C for 24hrs) but I have not been able to generate it. It has a C terminal 6x his tag and not being picked up by western blot. I am using pet24b vector.
I am new at these procedures and any help or suggestion from the community to shed light into this would be of immense help!
Thank you
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Codon frequency optimization for the target host is crucial in my experience. It can make the difference between no yield at all and decent expression, esp. when the phylogenetic distance is huge, as it is in your case, between a plant and a bacterium.
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hello everyone
We performed a peptide-Doxorubicin conjugation reaction. The mechanism of the reaction's conjugation is based on solid-phase peptide synthesis.
In a nutshell, we combined DOX, HATU, DIPEA, and FRRG [the peptide] in DMF to produce DOX-FRRG and a few other small molecules were also produced as by-products . Preparative RP-HPLC is the conventional method  used for purifying frrg-dox. I'm curious whether there are any other methods for purifying the solution, particularly cheaper ones.
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If your peptide conjugate is really a tetramer with sequence dox-FRRG, then it's not much larger or more hydrophobic than your impurities. So a SEP-PAK or desalting column may not provide enough resolution, although you can try.
You may be able to use ion exchange, to exploit those two R, and (presumably) capping the N-ter with dox. If you try that, maybe elute with NH4-OAc (volatile).
RP-HPLC is the gold standard for peptides. You apparently already have analytical RP-HPLC capability, to identify your reaction products. If you only need a few mg, you can combine a few runs on a 4.6 mm column.
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Hello everyone,
I'm planning on running my peptide samples on a high-resolution LC-MS instrument. I'm going to use a ZipTip C18 tip for the extraction of peptides and desalting of the sample. However, I'll be sending these samples overseas and it might take 2-3 days for them to reach their destination. I can potentially keep them in dry ice throughout their delivery, but it is very costly and we had few issues before where the dry ice evaporated until it reached the destination.
If I free-dry my peptide samples, do you think they are going to be stable for couple of days? Considering there won't be any humidity where the enzymes can work on the peptides, but I just wanted to get the opinion of people who has lots of proteomics experience.
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Selam Bora,
leave your peptides on the zip tips (after washing step without elution)and send them in an envelope at RT. The MS-facility can than elute the peptides from the C18 tips and start LC-MS analysis. I have done this several times with custom made C18 stage tips (much cheaper than ZipTips) in a collaboration with colleagues from Acibadem.
Let me know if you need further assistance.
Good Lick,
Murat
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I am synthesising the tetrapeptide, GPOG, following Fmoc strategy in the Solid Phase Peptide Synthesis (SPPS). After the final cleavage from the resin, I evaporate the cleavage mixture (TFA, m-cresol, m-thiocresol, water) in a rotary evaporator. Then I add cold diethyl ether to the evaporated peptide solution to precipitate the white peptide powder.
I then centrifuge the mixture (white peptide powder dispersed in diethyl ether) at 8000 rpm for 10 min at 20 C. In this process, the peptide powder changed to a light brown sticky mass. The yield of this mass is very less in comparison to the amount of white powder that I had first got by adding cold diethyl ether.
Can anyone please provide any explanation and solution for this?
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It could be the case, based on the quality of your ether ;-)
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Does anyone know how to do a Job Plot for the binding stoichiometry of peptides and cyclodextrin?
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Should I include the mole ratio on Y axis ?
I read some literatures that they plot it
abs* ratio vs ratio . But I did not get the same result as I plot it abs vs ratio .
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Hello Community,
I'm about to construct some peptide sequences ending in 2-azidoacetic acid. In the literature there are several papers with more or less standard procedures with this compound. For example:
Yu, M. et al. Inorg. Chem. 50, 12823–12835 (2011)
Lundquist IV, J. T. et al. Org. Lett. 3, 781–783 (2001)
However, none of those papers utilise 2-Cl-chlorotrityl resin. All use Wang. My question is WHY? Is there some reason that you cannot couple an azido-AA on a peptide using 2-Cl-Trt resin???
Thanks ahead for comments.
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It could be the case that the authors wanted the side chains to be fully protected after the SPPS stage. Also, what if the azide partially decouples the peptide from the resin?
Well I hope you find the answer and share it here ;-)
Best of lucks!
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Hi,
I am looking for approaches to grab short peptides (5-10 mers) other than a 1000 or 3000 MWCO spin filter, do you have any suggestion?
Thanks
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I suggest using Superdex 30 Increase SEC columns from Cytive which are designed for high resolution, small-scale purification, and analysis of small biomolecules with Mr ~ 100 to ~ 7000, such as peptides...
Good luck...
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I have a neutral peptides that is insoluble in pH 7.4 solvent. I need solution to be pH 7.4 for the subsequent reaction. I have tried DMSO to help dissolve the peptide, but it would be precipitate if I regulated the pH to 7.4. What can I do to try solving this problem?
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ACN as co-solvent
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I'm trying to build outer membrane of e. coli with small peptides above it, with CHARMM-gui. Peptides should be positioned above the membrane, but charmm-gui creates space for them between LPS (lipopolisacharides) pushing LPS-s to the edges of simulation box.
If I use only one peptide, it is not problem, empty spaces is not big, and charmm-gui sucessfully finishes job. But when using more peptides, empty space created underneath them is large, so LPS-s are pushed to the edges, tightly packed, which obaviously causes problem for charmm-gui and gives error:
"Charmm terminates abnormally. Please check the output or report this failure to the CHARMM-GUI developers. The bilayer generation is stopped to prevent an infinite loop. Please refresh the browser to restart the bilayer generation with different random seed."
I tried several times, with the same outcome. It looks like membrane builder thinks that peptides are "inserted" in LPS, so it leaves empty space for them in between LPS-s.
I report error to charmm-gui developers, and got short answer: "View “step3_packing.pdb”. "
I looked, and see nothing strange, nothing to lead me to solve the problem.
Any idea where to look, or how to solve problem?
Third picture is outer membrane without peptides
Thanks
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What about adding your peptides on the other side of the bilayer?
If you are using pbc they should still be able to move over and interact with the LPS. Just be sure to put a good bit of space between them and the inner leaflet.
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I am carrying His-Pull down Assays with purified peptides to investigate binding. I repeat the same protocol meticulously, prepare fresh imidazole every time I carry out a GST-PD, have BSA (0.5mg/ml) in the binding buffer, etc. I do have positive results so I know the assay works and that there is binding but repeating the assay gives me some or the other error such as non-specific binding with the negative controls. So, I really want to know if there's anything I can do to rescue the irreproducibility?
Also, I would like to know the usual method of normalizing the GST-PD data for binding.
Thanks
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Thank you So much John. Yes, I agree I wrote it the other way. It is His-Pull down and I am trying to pull down GST-peptides with it. Thanks for your suggestions
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How to remove N- methyl morpholine from the coupling reaction for getting a pure compound?
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Hi everyone,
We are currently working on the characterization of a complex protein mixture by MS/MS and when searching in the sequence database (transcriptome) with PEAKS X+ software. He does not seem to be able to assign peptides for sequences containing unknown amino acids (noted "X" in the sequence) neither with "*" were the software simply remove those sequence from the database.
How can we solve this problem, which annotation for unknown amino acid is required by PEAKS software ?
Thank in advance if someone can help us.
Damien
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Mr. Redureau,
Please, concentrate on the theory and model equations behind references [1-4]. Currently, only this approach can provide exact quantitative and 3D structural analytes mass spectrometrically, because of only it has absolute predictive capability in terms of chemometrics. It is applicable to complex mixtures of any analytes in condensed phases.
[1] Analytical Chemistry Letters, 10 (2020) 703-721; Stochastic Dynamic Mass Spectrometric Approach to Quantify Reserpine in Solution; Bojidarka Ivanova, Michael Spiteller;
[2] Steroids, 164 (2020) 108750 Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller;
[3] B. Ivanova, M. Spiteller, Mass spectrometric stochastic dynamic 3D structural analysis of mixture of steroids in solution – Experimental and theoretical study, Steroids 181 (2022) 109001:
[4] Journal of Molecular Structure, 1260 (2022) 132701, Stochastic dynamic quantitative and 3D structural matrix assisted laser desorption/ionization mass spectrometric analyses of mixture of nucleosides; Bojidarka Ivanova, Michael Spiteller, respectively.
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I have performed docking of a peptide epitope with MHC complex using Galaxypepdock software and have obtained 9 different models along with protein structure similarity (TM score) ranging from 0.970 to 0.995, Interaction similarity score (220-250). How to go about the analysis as I am not able to see my peptide docked with the protein structure?
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You can visualize your result using Discovery studio or Pymol.
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Hello,
I am very new to the FTIR technique and recently stumble upon an observation that I cannot explain.
So when I analyzed the native proteins, the spectra showed no transmittance in the Amide A range. But when the protein was hydrolyzed first prior to measurement, it showed a peak in Amide A range.
From reading several books and publications, I understand that Amide A corresponds to the bond between N and H in peptides. So why does it require hydrolysis of a protein into peptides in order for this peak to show?
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Hi,
Considering the fact that FT-IR is indeed sensitive to the higher order structure of protein molecules, it could be that certain vibrations of bonds do not appear in the intact protein, since an NH group might be involved in a beta-sheet, but does become apparent when that secondary structural element is lost upon digestion of the protein, which eradicates most of its higher order structure.
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Even though I have used very little concentration of p-NPA, saturation has not been attained for 5000 seconds. But researchers have done a kinetics study of peptide catalyst with the same concentration of substrate i.e., p-NPA (that I am using for my work), and monitored catalysis for 400 seconds. But, I am unable to attain saturation even within 5000 seconds for 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM and so on. So, if saturation is not attained, then how to calculate the value of Vmax, Km, Kcat? Can anyone kindly suggest to me, if I am doing anything wrong at any step? It would be really helpful.
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@Kalpana Kumari...
Everything seems to be right...
What kind of model you plan to use...to find Kcat and all?
One way is to use Michaelis-Menten equation to determine the Km and Vmax...
How do you calculate basically?
One more thing...at what temperature and how much time(maximum) you wait for it?
If something you feel confidential...you may directly message me.
Thanks and regards,
Aadhityan A.
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I am looking for a tool (online, R, Python, or otherwise) which I can use to highlight peptide sequences on the full protein sequence in a visually nice way for publications and presentations.
Extended description: In several of my bottom-up proteomics research projects, I have identified proteins of interest for a given condition/disease. Often, these proteins are activated/deactivated by cleavage (e.g. the complement system, coagulation system, angiotensinogen, etc.). Therefore, I commonly perform a peptide-centric analysis after the protein centric analysis, to identify changing peptides and then I manually map these to the protein sequence. I am looking for a tool to help me with this; where I can submit the list of peptide sequences and have these visually mapped to the full protein sequence of origin. Ideally, the tool should include known cleavage products (e.g. from UniProt KB).
Any advice is most welcome and thank you for your time.
Sincerely yours,
Tue Bjerg Bennike
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Maybe Peptigram can be of use for you?
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I am wondering if my proteins are digested into small peptides, will the protein concentration remain the same if I use BCA or Nano-drop to detect the concentration.
Many thanks.
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It's an interesting question. BCA is based on copper reduction which is promoted by particular side chains, and also the peptide backbone to some extent. Peptides can be quite small and still react in the BCA assay (tripeptides are said to be large enough, but not amino acids or dipeptides). On the whole, you are likely to get peptides that are long enough in a digestion experiment, hence there may be little impact on signal.
In the case of Nano-drop, you presumably mean measuring absorbance at A280? Here the aromatic chains generate most of the signal (particularly tryptophan) and these residues would still be present after digestion, no matter how extensive.
Obviously you are adding protein to the system (trypsin), and you would need to control for that, though normally the % would be quite small.
Of course, it would be better to do the experiment +/- trypsin to be sure.
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To provide some context:
Once an antigen is processed endogenously or exogenously by the APC's (ex: dendritic cells), it is then presented on MHCI or MHCII as a peptide.
specific naive Tcells have a matching Tcell receptor that is complimentary to the presented peptide. considering the incredible diversity of peptide antigens that can be presented, what governs the production of Tcells having the exact match to the presented peptide?
What is the mechanism of Tcell receptor diversity considering that somatic hypermutation is designated to Bcells?
(picture adapted from: Molecular biology of the cell - Garland science 2008)
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It is stochastic, or random. The receptor repertoire based on vdj, alpha beta v delta gamma and n and p additions result in a nearly unlimited. Umber of potential receptor rearrangement. The naive t cells enter secondary lymphoid organs at the instruction of chemokines and 4andomly test against antigen presented in the context of correct mhc. In this scenario, it I'd the antigen that selects the correct tcr, resulting in clone proliferation. The t cell is the passive player.
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Can I use MPLC chromatography to test Oligonucleotides, Peptides and Microbial-derived proteins
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Hello,
I am studying protein corona formation and composition on coated gold nanoparticles. I coat them with polymers and incubate in FCS or plasma. I now want to compare between differently coated particles what relative amounts of proteins are bound. I don't need nanograms, it's enough to say "Particle A binds twice as much BSA/protein X as particle B".
I use MaxQuant to process my Orbitrap data and I can see what proteins bind in general. But how can I quantify and compare? I read about the Top 3 approach. Did I understand it correctly? I should add a known amount of a reference protein (they use BSA mostly, but I would need to use something else, because BSA is a protein of interest. Any suggestions?) and then they use three peptides of a protein that show the highest intensities, add the intensities up and compare it to the reference?
How do I know which peptides belong to which protein? Do I need to do that all manually in an Excel list of like 300 proteins? What kind of reference protein should I use? Does every protein have at least 3 peptides? Would I need to look at unique peptides only? It's quite hard to find a detailed explaination on this.
Thank you very much for your support!
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Hi Julia,
I have performed the Top3 analysis several times to compare the quantities of different corona proteins within one nanoparticle and also between different nanoparticles. It is a lot of work to do it with excel and you need to have good excel skills to do Top3 analysis with excel.
From the Maxquant combined/text folder you need to get the info for all peptides assigned to the identified proteins from the evidence.txt file where you need to perform a lot of sorting and filtering for each sample individually and finally need to do the alignment for the Top3 values of each protein in your list to compare the different samples.
Was the MS analysis performed by Benno? Then he can possibly help you with the Top3 analysis. If not let me know so I can try to give you a step by step procedure or we can do a zoom meeting were I can show you what to do with the data to get the required Top3 values.
Best,
Murat
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Dear all,
I am doing an MD simulation of a peptide with 3 unnatural amino acids and a linker using Gromacs. I have already generated the itp files for unnatural amino acids and the linker using ATB web server.
Can anyone suggest to me how to generate topology(.itp and .top) for the whole peptide chain ?
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It depends on the geometry of the H atom to be added. For example, if you want a planar HA lying in the plane of atoms (CA, -C, CB), then the entry in the hdb file would be like
; res # additions
XYZ 1
; Number of H add H Type H i j k
1 1 H CA -C CB
Here -C means a common atom to whom CA and CB are bonded to create a reference plane.
For more info on different H-Type to be generated, please follow page 421 of the following Gromacs manual
I hope, this helps you to create a hdb file for your molecule.
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Are there any methods/techniques to detect the peptide present on a MHC molecule? If yes, please let me know how it is done.
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I am trying to purify a hydrophobic peptide (A-beta 42) that is 4kDa in size. So, I am using reverse-phase chromatography equipped with a C18 column (1.7 um). I am expressing the peptide in BL21 cells and I have confirmed that the induction was successful by running the cell lysate on a tricine system SDS gel. However, I don't see any peaks on my chromatogram when I run the total cell lysate. I tried to run a commercially available sample of the same peptide dissolved in water and I can clearly see the peak on the chromatogram. I am not sure why is that... so I reran the same sample on a gel, and I can still see my cell lysate present in the sample. Is there any suggestion to troubleshoot this... I am thinking to add His tag and use the affinity column but I see that most papers use C18 columns...
I am attaching the chromatograms of the sample and the standard for reference.
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1) Try looking at your chromatogram using a log(absorbance) scale. The peak you are trying to see may be below the threshold you have set for automated peak detection. If you are certain that the abundance is high via SDS-PAGE, then:
2) It is probable that the peptide of interest is binding to another protein in the lysate and won't resolve independently in the solvent system your are running in the LC. In a gel, you are adding SDS to denature all the proteins and solubilize the hydrophobic proteins in an SDS coating. You won't be able to run SDS on a C18 column, however, you might be able to find alternative chaotropic solvent systems. I would suggest high urea or thiourea as a start, these can be tricky in LC columns as you approach saturation. You may have better luck with non-detergent sulfobetanes (NDSB). Another alternative is 50% acetic acid in water, as long as your column can tolerate it.
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I want to fuse the P2A DNA sequence between 2 genes of interest. Can the P2A peptide have the ability to self excise when the polpeptide chain is synthesized in prokaryotes?
Most examples for this procedure are typically done in eukaryotic system. I am not sure if this ability of self excision would happen in prokaryotes? Maybe someone has/have experience(d) this phenomenon.
Any ideas are most weclome, if any :)
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This is unclear - however, 2A peptides are thought not to induce self cleaving per se (wherein you might expect it to behave the same way in either organism), but instead rely on a ribosome skipping mechanism, in which it skips over this sequence and produces two separate proteins.
This might be a feature unique to the eukaryotic ribosome, so it is possible it does not work in prokaryotes
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My peptide is highly soluble in water.
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Dear Suresh Rajamanickam thank you for posting this very interesting technical question on RG. DIPEA (= N,N-diisopropylethylamine) has a relatively low boiling point of 127 °C under normal pressure. This is not very different from the boiling point of water (100 °C). Thus I would suggest that you dissolve your crude peptide in a small amount of water and evaporate the solution to dryness under vacuum (e.g. using an oil pump). Perhaps at the end you can slightly heat the residue (ca. 40-50 °C). That way you should be able to remove the remaining DIPEA together with the water.
Good luck with your work and best wishes, Frank Edelmann
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I use 3 peptides, I dilute them in Dh20.
When I put in the media after 3 hours my peptides precipitate in the cell culture media.
When I remove the serum is possible to avoid it. But I need to put serum in my cells.
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The only thing I can suggest at this point is adding single components to DMEM with your peptide to see which one(s) is causing it to precipitate.
Hopefully, someone with more peptide chemistry or laminin peptide experience will come across this thread to give you a solution.
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How will it affect the conformation of peptides?
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Dear Kalpana Kumari in addition to the expert answers provided by John Carter please also have a look at the following potentially useful article which might help you in your analysis:
1,1,1,3,3,3-Hexafluoro-2-propanol and 2,2,2-trifluoroethanol solvents induce self-assembly with different surface morphology in an aromatic dipeptide
This article is freely available as public full text right here on RG. Good luck with your work!
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Hi. Is it always invalid to get the docking result obtained using structures that have not undergone energy minimisation? What would be the factors considered in this decision?
YASARA Structure allows the users to build the missing residues in protein crystal structures using BuildLoop and OptimiseLoop command. If the missing residues are modelled using BuildLoop and OptimiseLoop command only without energy minimisation, is the subsequent docking result valuable and valid. In other word, should the data generated this way should be discarded as invalid?
Alternatively, is it meaningful to retain the docking results generated using the same modelled structures with and without energy minimisation, and hypothesise that the best-ranked peptides obtained in both methods can be one of the best peptide inhibitors, which can only be confirmed experimentally?
Thank you.
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Yes, it is mandatory to determine the proper molecular arrangement in space since the atomic coordinates of the protein structures are not energetically favorable. . The aim of energy Minimization is to find a set of coordinates representing the minimum energy conformation for the given coordinates b/w the amino acids. Lower the energy more stable they are.
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I have been running a förster resonans emission transfer (FRET) assay, using a dnp/mca conjugated peptide probe for my protease of interest. However I have problems with the assay; sometimes the assay works fine and I get really nice and clear signals, but sometimes the signal from both the FRET-peptide control (w/o protease) and buffer only control (i.e. only the buffer) declines dramatically during the timecourse of the experiment, and the signal from the protease is much weaker than normal (after correction for the declining background...).
Experimental setup:
Peptide: dnp-NH-GTQVKLIGHR-CO-mca. Detected fluorometrically at 392 nm using an excitation wavelength of 325 nm according to litterature. 20ug/ml final concentration
Buffer: 50mM Hepes (pH 7.4), 200 mM NaCl, and 2 mM DTT
Enzyme: Ctss, 0.1 ug
Plate and buffer pre-heated to 37 degrees.
Read in Spectramax M2e using fluorescence kinetik read with 325 exitation, 392 emission and 325 auto-cut off filter.
Any idea of what might be going wrong?
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I can think of two potential problems, both with simple solutions.
1. The peptide is sticking to the plastic of the microplate. The enzyme can also do this. The solution is to include in the buffer 0.01% Triton X-100 or some other non-ionic detergent at a concentration below its critical micellar concentration.
2. The fluorophore is suffering photodamage as a result of an excessive number of flashes from the light source. There are two simple solutions. One is to reduce the number of flashes per measurement. The other is to make measurements at less frequent intervals.
Make sure you always use the same settings for the measurement for every experiment in order to get consistent results. You can save the setup to prevent accidentally using the wrong settings.
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Hello
Im using a known peptide sequence of gene of interest from phytozomw to tblastn with SRA data in NCBI. My sequence results doesnt cover the query and arent highly similar.
After downloading the fasta sequence of all the aligned hits I blastn it but my plant of interest is not in the results.
What can I do to find the desired sequence for my plant?
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The above may be helpful. If NCBI does not give significant similarity, I would interpret in the database there is no similar sequence to your protein of interest. Another way includes a "try and error" to find domain similarity, functional similarity or others, but you will need to explore a different approach. Good luck.
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I have a peptide with non standard amino acids and their parameters are in CHARMM36 format (.prm and .rtf). moreover, the parameter file contains a PATCH to add a thioether bond.
Is there a way (other than CHARMM-GUI, because it cant apply a custom PATCH in the uploaded files) to prepare the files manually for GROMACS while applying the PATCH?
Thanks
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The most feasible way to create topol and itp files for a new residue and ligands for molecular simulations with GROMACS is using acpype and antechamber. The workflow starts with the parameterization of electrostatics (atomic charges) with some (S)QM methods, after which the other parameters (bond stretching, angle bending, torsional, and vdW parameters) are grabbed from pre-fitted general force fields (e.g., GAFF). After the generation of the parameters to describe the new specie, the parameter files would be transformed into AMBER, GROMACS and CHARMM formats for later use in the construction of your simulated system.
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Hi fellow RGers, in my experiments, I'm trying to express a peptide and I'm curious about the general stability of the expressed peptides. Is there a rule of thumb concerning peptide stability in bacteria? I have heard from other colleagues that peptides are generally more stable when it's secreted vs in the cytosol. However, I cannot find any papers confirming this. Have anyone came across any data in literature that supports it? Thank you and have a nice day.
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Thanks
Pierre-Marie Andrault
for weighing in. Will take the note on BL21 if I'm expressing my peptide in E. coli. I was trying to express a peptide in Clostridium and I could detect it with ELISA in the supernantant as opposed to the cytosol so it seems the saying is correct
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Hello, I am interested in working with dyphenilalanine but I am having a hard time identifying the correct item on Merck or other site to place an order. Specifically:
  • Small diphenylalanine, NH2−Phe−Phe−COOH (FF), peptide monomers from the work Strong Piezoelectricity in Bioinspired Peptide Nanotubes, Kholkin et al 2010, I have not been able to identify this one anywhere
  • For L-Phenilalanyne from Simultaneous Synthesis and Self-assembly of Cyclic Diphenylalanine at Hydrothermal Condition, Togashi et al 2006, it this it https://www.sigmaaldrich.com/PT/en/product/aldrich/s452777 ?
I would appreciate it if anyone could guide me towards the correct peptide as well as help me understand how to correctly identify similar things in the future.
Thank you in advance.
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Hi Carlos,
Thank you for you reply. I was able to contact the authors of the first article I mentioned where they use NH2−Phe−Phe−COOH for nanotube synthesis. Although what I am really looking for is diphenylalanine for microsphere synthesis which is shown in the second bullet point's article. I am unsure if the L-Phe mentioned is the same I linked to Sigma's product page, would you be able to confirm or deny it? Also, do you know if L-Phe is piezoelectric?
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my peptide molecule having free acid and gunidine group ,How can we crystallize and what are the best solvents for crystallization ?
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Hi. I am currently designing peptide ligands.
I noticed that DUD-E (Directory of Useful Decoys, Enhanced) can be used to generate decoys for small molecules. I wonder if DUD-E is equally useful in generating decoys for the peptide ligand.
Is there other way of decoy generation for peptides?
Thank you.
Sincerely yours,
Thai Leong
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A good decoy would be randomizing the sequence of your query peptide, an construct some of them. This would give you a set of same length peptides with the same aa composition. This is similar to the procedure in BLAST for estimating E-values for alignments
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Hello, does anybody use the OPA reagent to quantify peptides by fluorescence?
I'm struggling with the conditions, specially with the ratio between OPA/peptide to do the calibration curve. I follow the Thermo Fisher protocol but it's not working. Thanks in advance
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Hey Maria De Los Angeles Ramirez , is there a way to use HPLC? Check this paper for a method DOI: 10.3920/JIFF2021.0017
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related to Cytotoxicity and cell viability
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Nanomaterials can interfere in assays through quenching of transmitted light or fluorescence. Therefore, careful consideration must be given to fluorimetric/colorimetric in vitro toxicological assessments of optically/chemically active nanomaterials in order to relieve any potential artifacts due to the nanomaterials themselves.
Please refer to the article below for more information.
I would recommend the use of MTT assay. Please refer to the review article below.
Best.
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Our primary hippocampal cells are simply too happy in the presence of Abeta1-42. While the neurites are definitely destroyed in the presence of micromolar quantities of oligomers (prepared with the standard method, ie resuspension of denatured film into DMSO to 5 mM, sonication, then dilution/incubation to 100 uM in cold Ham's/DMEM overnight at 4C, and spinning before application), 4 days after receiving micromolar concentrations of oligomers, the cells still give the same viability response using WST-1 or CCK8. We've tried two peptide sources. Has anyone solved this problem?
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Iris Lindberg We have same question now,can you provide your catalog number of Abeta peptide?
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I am currently working on expressing and purifying recombinant strep-tagged AMPs peptides in plants. My usual strategy is after whole protein purification, I do strep-resin based strep-tagged peptide purification, then perform on-resin digestion with the protease followed by His-Trap purification to get rid of my His-tagged protease, then size-exclusion chromatography with the column for resolving peptides lesser than 3 KDa. After that I finally perform RP-HPLC using C18 columns to finally purify my peptide and concentrate it.
I have heard about flash chromatography for peptides and I was wondering for purification, I can do Strep-Resin based peptide purification followed by on resin digestion and then go straight for RP-HPLC bypassing the His-trap and Size-exclusion chromatography step? Is it a feasible option to do this as I am going to purify 1 gm of peptide and I am trying to find a good and less time consuming strategies but to get good quantity and quality peptides.
Any suggestions on this would be most welcome.
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The most widely used method for protein purification is affinity chromatography, which separates proteins based on their specific interaction with a matrix. It is one of the most effective techniques, since it takes advantage of the incorporation of a structure of choice (called a tag) onto the protein.
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Are structure-based peptide drug design and ligand-based peptide drug design mutually exclusive? If we have the crystal structure of the protease and design the peptide inhibitor based on the sequence of its ligand (protease cleavage site), is it a structure-based peptide drug design and ligand-based peptide drug design or a hybrid of both? Thank you.
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Dear all,
I need to synthetize a peptide for antibody production, since I had a hard time to purify one very stubborn protein.
Please, help me to figure out the right strategy how synthesize a peptide from the scratch?
Thank you!!!
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DEAR Oleksandra Kepple
Peptide synthesis is the process of making short sequences of polypeptides by adding one amino acid at a time. This process is useful for creating specific sequences that represent epitopes of certain protein domains that may or may not be modified by moieties, such as phosphate groups.
Peptides are synthesized by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. ... Chemical peptide synthesis starts at the C-terminal end of the peptide and ends at the N-terminus. This is the opposite of protein biosynthesis, which starts at the N-terminal end.
please consider this link:
Syntheses of Peptides. The First Epoch
  • January 1991
  • DOI:
  • 10.1007/978-3-642-75850-8_2
  • In book: The World of Peptides
  • GOOD LUCK
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Good morning,
I should label peptides with cyanine dyes, which are very unstable molecules. Do you have any advice to handle and purify the product?
Thank you,
Margherita
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In addition to what Guobing Xiang has written, store the active ester in aliquots desiccated at -20°C or colder, prepare fresh solutions for each labeling experiment and use the solutions immediately. Don't use buffers with amino groups, as they'll happily compete with your protein for quenching the reactive dye.
If you should need to do a clean-up after the labeling, use an as small as possible amount of gel filtration material (e.g. a Sephadex G25 based column) and use as much protein as possible (ideally at least 5mg), as you might lose a lot of material when you start off with a few 100 micrograms.
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Hi,
I'm trying to measure the concentration of my pure peptides with Thermo Scientific NanoDrop One. The peptide sequence is ELAGIGILTV, which does not have the key amino acids required for measuring proteins at A280, so I measured the concentration at A205nm instead. However, based on the readout from NanoDrop, the peptide absorbs higher at A280, and the concentration measured at A205nm is also unreasonably low.
Does anyone know the general rule of choosing the proper program to measure peptide concentration?
Thank you!!!
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Well, it's good that you have seen the magnitude of the DMSO signal. Although you can make it 'disappear' by blanking there will be a (unknown) % error on the measurement prior to blanking. For example, if you read DMSO vs water blank three times you could easily get 700, 703 and 698. You might have chosen to blank on any one of those readings, giving potentially three different zero reference points, even though they are all 'zero'. If the reading due to peptide + DMSO is anything like the DIFFERENCE seen between the readings made on DMSO solvent alone vs water blank, then you potentially have a massive % error on the peptide reading. Let's say you got a reading of 5 at A205 for peptide + DMSO. If the (substracted) reading for DMSO was wobbling in the range 698-703, it could make a huge difference. Generally, subtracting a large reading either mathematically or by blanking the machine is not good practice. Your measured value needs to be substantially greater than any variation in the system.
If DMSO is causing high signals at A205 (and it could be impurities, not just the DMSO), it would be better to use a more UV transparent solvent. If the peptide in DMSO can be transferred to water (perhaps to give 10% DMSO final conc) your readings may be easier to make, though the same principle applies, the measured value for peptide has to considerably exceed the errors elsewhere in the system.
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I have peptide sequences. I want to add it into a software to predict whether it has anti-microbial potential.
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This web site may have what you are looking for:
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i have a peptide mixture ( bought from a company) and i would like to purify it
The sequence is Acetyle-KCYCSIGGGTRQCYATLAECR-NH2
i expected to have 2 disulphide bonds in this peptide
i would like to purify it using C8 column(9.4*250mm).
I dissolve the sample in 90% Water and 10% ACN in 1 millilitre and filter it .
Mobile phase ACN with 0.1 TFA
Water with 0.1 TFA
Flow rate :2 ml/min
Injected volume : 20 microliter
detected at 3 wavelength : 210nm -220nm -280 nm
Gradient
Time Water ACN
0 87% 13%
5 87% 13%
35 40% 60%
45 40% 60%
50 87% 13%
i collected all the fractions and diluted it 10 times for ESI MS .
but i do not see the peptide with all peaks i collect in ESI MS (find attached of chromatogram i got ) . Any ideas please
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What about the peak with the retention time 15.486 min? Tyrosine residue justifies absorption at 280 nm. You did not catch that peak for MS, if I understood well your description.
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Why cont we use negative ESI for peptids
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I agree with Andre Fabris. ESI is a mild/soft ionization technique that performs ionization mainly by transferring or removing hydrogens. This typically ends as detectable hydrogen adducts of the molecule at positive mode as precursor ion. Besides other adducts, this formation needs proton acceptor-bearing molecules such as peptides. Basic amino acids such as lysine arginine histidine and other nitrogen-carrying side chains of peptides make it more prone to accept hydrogens thereby ionization at positive polarization.
Take a look at this for fundamental,
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Hello, Research Community (RA),
I am looking to use a Pepsite2 bioinformatic tool for computing the binding residues of receptors (ACE) when used with food peptides. Please let me know if the enzyme model is required to prepare for that. I mean to say this tool asks for the PDB code and chain id of an enzyme that can be easily found from the protein data bank. But is there any treatment needed for this PDB structure like removal of a lisinopril inhibitor or water molecules?
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Dear Sonu Sharma , i m not 100% sure about the preparation process for pepsite2 for the second uploading option. If you use the first option where you enter the PDB code you don't need any preparation, you can see in the results that they have removed the inhibitors, but no idea If you add the PDB file manually, I advise you to try the second process with a protein that had been already processed with this software, take anyone from the literature. I wish you the best.
kind regards
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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.