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Protein-Protein Interaction - Science topic

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Hi All:
I am trying to purify an endogenous protein. But the protein is known to have many other interacting proteins in the cell system I am working on. Is there any way I can decrease protein-protein interactions during IP, remove its interacting proteins, and purify my protein of interest alone? Thanks a lot for your help!
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A high salt concentration may be helpful when electrostatic interactions are involved. A detergent may be helpful when hydrophobic interactions are involved.
Some interactions are not strong enough to persist when there is a high degree of dilution, as in immunoprecipitation, with all the washes.
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Dear all,
I tried docking my protein heterodimer to form a hexamer (trimer of a heterodimer) using symmdock server. The output results has given top 20 models of the hexamer and I opened the top model into PyMOL. I see the the parental template has intact structure while the generated partner structures are all broken in the PyMol (Picture attached). When I opened it in coot all the atoms are present and display very well as atoms. But somehow it is messed up with the cartoon representation in PyMol. Please suggest, how can I fix this issue.
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You need to make sure from the beginning (before docking) that the "receptor" and the "ligand" do not have the same chain label! You can change chain labels using the "alter" command in PyMol ( https://pymolwiki.org/index.php?title=Alter&redirect=no )
alter (ligand and chain A), chain='B'
changes the chain label in object Ligand from A to B
You can force a proper display of your faulty complex by using the "retain_order" setting in PyMOL ( https://pymolwiki.org/index.php/Retain_order )
set retain_order, 1
but the ambiguous chain labels may cause problems with a number of other commands and further evaluation of the complex in other programs!
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We are looking for receptors in order to perform in silico studies of some antalgic and anti-inflammatory ligands isolated from plants and have been tested in vivo.
A part from COX 2, IL-6, TNF-alfa, could anyone propose other important receptors?
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You can go for NF-KB, IgA IL-1, Toll-like receptors, RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors.
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Hello, all! I'm performing an experiment in which I'm doing a CoIP in the presence or absence of RNase to see if a protein-protein interaction is dependent on RNA. I am trying to identify a protein pair that is known to interact in an RNA-dependent manner, particularly in mouse embryonic stem cells (mESCs). I was thinking of trying to identify some proteins involved in translation initiation or spliceosome assembly/function, but am unfamiliar with the biology of these complexes.
Any and all help is greatly appreciated!
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In case anyone is curious about this question, I've found that the U1 snRNP complex components U1A and U1C interact in an RNA dependent manner and give a great Western blot signal. I'd recommend them as a control for RNAse degradation.
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I'm learning about the role of the centromeric histone H3 (CENH3) in the loading and assembly of the kinetochore complex. In many articles, light is being shed on the binding affinity between CENH3 and other major proteins in the kinetochore like CENPC and MIS12. I am looking for a software that could convert protein-protein affinity into numbers in order to be able to compare which protein of the kinetochore will more likely binding CENH3 than the other, and, if so, will these numbers show any correlation with publication.
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Dear researcher
Please consider this paper for protein-protein affinity prediction based on sequence:
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What exactly is the role of HSP-90 in extracellular environment of the cell? I am wondering whether hsp90 is involved in the translocation of the client protein from outside to inside of the cell. If somebody is having some references please share with me. I am very curious about this molecule.
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My article about that just got accepted you can see it soon, including tests before, immediately and 2 hours after exercise
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Hello,
I was wondering if someone has ever tried to use the BioID technique to identify extracellular interactions such as ligand/receptor interaction?
Thank you in advance
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Thank you for your response and sorry for this very late feedback.
For those who may interested, fusion of your P.O.I with HRP appears to be usable for such experiments as it work at pH compatible with extracellular proximity labelling with biotin.
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I am aware that TiO columns are used to enrich phosphorylated peptides after digestion of proteins, but can they be used to purify full phosphorylated proteins from crude cell extracts? Something around 30 kDa
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Whereas It is possible
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I want to know the expression change of my target protein by western blot from the control and drug-treated cells. 48hrs. after the treatment, I have pelleted the cells and extracted the proteins using RIPA buffer (50mM Tris-HCl pH 7.6, 150mM NaCl, 2% NP-40, 1% SDC, 0.1% SDS, 2mM EDTA). After denatured the proteins using SDS-PAGE loading buffer, I have loaded the samples in SDS-PAGE wells. But, some of the samples leaked from the resolving gel and it did not stack properly. The samples spread laterally as soon as they entered resolving gel. I have prepared fresh RIPA also, but the result did not improve. As a control, I have run my old extract in the same gel. but it did not leak. My labmates use the same reagent, but they do not face any problems. Previously I have thought that it may be due to sample overloading so I have reduced the volume. But it did not seem the actual cause. It would be very helpful if anyone inform me of the root cause of this type of sample spreading in the gel. Please find the images, attached below.
Thank you
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SDS-PAGE gels have a limited shelf life, in an air-tight container wrapped in wet tissues about 2 weeks. Disassemble a casette to check that the gel is firm and well formed.
Also, I'd reduce the salt concentration in the homogenisation buffer to, say, 50 mM.
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All extant protein alignment algorithms that I am aware of perform poorly for complex proteins that have many possible isoforms. There are better ways of making these assignments. Do you know of a new approach?
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I have made a protein-protein docking and performed simulations using Gromacs. I was to make MMPBSA study on it.
1. The docked complexes residues are not in a continuous sequence. (eg. Chain A has 1 to 100 and Chain B has 1 to 90).
2. The atom numbers were sequentially arranged. (eg. Chain A has 1-2000 atoms and Chain B has 2001-4000 atoms)
3. So I have created two separate ndx files with chain A and chain B separately. Now how to proceed with MMPBSA analysis?
I am following https://rashmikumari.github.io/g_mmpbsa/ tutorial. But how to provide two index files in Gromacs?
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Hello. Just open the gro file, look at the atom range and create single index file with two groups as Keerthic Aswin suggested. To do this type following command;
gmx make_ndx -f md_100.gro -o filename.ndx
a 1-3295
Press enter
a 3296-3431
Press enter
q
Here you go...and run this file for analyses. Hope this would be helpful.
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I would like to see if there is any interaction between my target functional recombinant protein expressed in high five cells and another lysate. Would it be feasible to combine both cell lysate solutions in one tube and pull down the possible interaction complex? All protocols that I found only use one lysate for Co-IP analysis. Has anyone done this before?
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I haven't done it before, but it seems like a reasonable experiment.
Another approach would be to purify the overexpressed protein , then use it to prepare an affinity resin. The affinity resin could then be used for the pulldown experiment with the cell lysate.
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I have docked two interacting proteins and get a pdb file, now I want to know the prediction of binding residue sites by this docked pdb file. Please suggest me some bioinformatic tools for this.
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Thank you, Diptendu.
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Dear fellow researchers,
I was wondering if different forms and stages of post-translational modifications (such as N-linked glycosylation) on a recombinant protein expressed in insect cells can be responsible for various thick bands observed post affinity purification* in my SDS-PAGE gel? I would need the unknown protein (predicted at ~55kD and possible glycoprotein) for interaction assays.
*Binding and wash buffer was 100mM Imidazole, 20mM Sodium Phosphates, 0.5M NaCl and 0.1% Tween-20. Elution was done with 500mM Imidazole and all the other components listed above. HisTrap 5ml column used.
I would be happy for any advice, opinion or past-experience.
Thanks,
Julian
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A likely explanation for the multiple bands is that the metal affinity chromatography was not sufficient to purify the protein to homogeneity, and the other bands are contaminating proteins.
You could check this by Western blot using an antibody specific to your protein.
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During Elution of protein (say an Antibody) from Protein A column using Elution buffer which is at low pH (3 to 4) and immediately adding Neutralization buffer to the collected fractions to prevent the negative effects due to this extreme low pH. I'ld like to know during this process will the protein remains in native form only (or) It will first unfold during low pH elution fallowed by re folding during neutralization ?
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Hi there,
Neutralizing the pH as quick as possible limits the denaturating effect of low pH on proteins. It's not about denaturation/renaturation processes, it's rather about preventing denaturation.
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I am currently working on Lactobacillus salivarius for an eventual bacteriocin production, I don't adjust the pH of the supernatant when realizing the deep well diffusion agar test. To check wether the prior inhibition effect of the supernatant is due to pH or to other inhibitors in the supernatant, I prepared a control of MRS-Broth-HCl solution at a pH of 4 that I tested against the same bacteria. At the same time and same plate, I tested again the supernatant that I have also at pH 4 , and surprisingly I got an inhibition only for the MRS-HCl and nothing with the supernatant, even though it is by pH 4 . Do you have any suggestion that may help ? Thank you very much !
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Could someone tell me how to download signed PPI (Protein-protein interaction) networks? That is, each edge is associated with a positive value (i.e., activation) or negative value (i.e., inhibition). I am trying to apply a new dense subgraph model on signed graphs.
Really appreciate your help!
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Perhaps you may check a few replies in this post:
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I'm studying negative sense RNA virus virus fusion with host membranes. But I don't have much understanding about protein folding and unfolding, conformational stability. I need detail about this concept.
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maybe this publication will be useful to You
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Hello there, I'm designing an experiment to investigate the different interactome of a protein across a range of temperatures, from room temperature to 40°C.
I plan to engineer the drosophila strains to bear C-terminal FLAG tags in this protein. After extracting cell lysate, I would use anti-FLAG antibodies to purify them (affinity purification), followed by mass spectrometry. This is repeated at different temperatures to see what proteins binds to the target differentially between normal and heat shock conditions.
The problem is, I couldn't find any related literature illustrating whether anti-FLAG antibodies still function well at higher temperatures.
Given the requirement of this research (to be in vivo, for instance, the cell lysate), i reckon affinity purification-mass spectrometry to be the most suitable way. Yet I'm a bit stuck now. Could anyone give me a few suggestions here?
(Your sharing of knowledge will be greatly appreciated!)
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Yes- your anti-FLAG antibody strategy may work at 40 degrees. However the comments on biology are also relevant. You could consider doing this in a thermophile that tolerates different temperatures. You could also use something like Green Fluorescent Protein (GFP) instead of flag as your tag and then use a GFP nanobody for pulldowns as then the GFP color will allow you to see your pulldowns and follow possible unfolding.
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Dear All,
What would be the best strategy to optimize a certain crosslinker concentration for my protein interaction studies?
Thanks
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My approach would be to test a wide range of crosslinker concentrations, keeping everything else the same. Use SDS-PAGE to examine the results. The most meaningful results come from the low crosslinker concentrations. High concentrations are more likely to result in artifacts, including intramolecular crosslinking.
The next step would be to fix the crosslinker concentration at the low level found in the first step, then vary the protein concentration, which will help you get an idea about the dissociation constant of the complex.
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I docked two proteins through ZDock server. Now I want to simulate this complex using Gromacs. I need some help. Which protocol do I have to use?
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I ran the MD as a protein, but now I am wondering how I can analyze the RMSD of the peptide after MD, there is a way to separate the peptide from the rest of the protein and calculate trajectories? ...Thanks in advance for your help
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I am working on a protein drug delivery system mediated by hydrogel as a vehicle. Even though the formulated hydrogel exhibited more than 2000% swelling, it could not able to deliver the protein drug. Scanning Electron Micrograph shows the hydrogel has a non-uniform porous structure. However, the average pore size is larger than the size of the protein drug.
I suspect that, there could be some chemical bonds (e.g. hydrogen bond) formed between the hydrogel polymer groups and protein as there are hydroxyl groups present in the polymers. Is there any way to find out the possible interactions between the protein and hydrogel? If there are interaction, please suggest on how to prevent these interaction? I am avoiding the use of extreme pH in the process as it may degrade the protein which is stable at pH 6.5-7.5. Thank you very much.
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Dear Ruben Chandran, since the gel is not specified, and if it is a matter of strong secondary interactions, then you you have to disturb the structure (breaking) either by changing either the pH or playing on the ionic strength by addition of salt. This way, mainly H-bonds will vanish, and see if there is drug relase after. My Regards
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Hi every body
I have a pdb file of a single domain protein(it is a truncated form of a two domain protein). this mutated structure express mainly as inclusion body and i want doing protein-protein docking to investigate that if the truncated protein does have strong affinity for binding to the same other truncated protein or not.
for that i want to use HADDOCK can any body helpe me with:
1-which tools is better for my goal?
2-how can prepare a pdb file of two truncated protein ?
3-should i define box and TIP3P and ions for my pdb file?
4-how can i remove segid from my pdb file and i dont know if my pdb file has this parameter or not.
i am very grateful if any one can help me with even one of these questions .
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Hello dear Golnoosh. Items such as the removal of heteroatoms, the correct definition of some PTM such as structural glycosylation in the structure are important for docking in the haddock. The server itself provides a file that explains how to properly prepare the input files.
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The concept of using the innate UPS system is very interesting and new to me. It seems like most PROTAC E3 ligases consist of ligands for cereblon. If I were to use a different E3 ligase other than CRBN that's known to be expressed in only certain tissues of the body, would my target protein for degradation be limited to that same tissue?
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Great question! Unfortunately, it's very hard to tell that in advance, and there are several pitfalls you should not forget about. Developing this (high-affinity) PROTAC is only the first milestone. Then you have to make sure you can administer it in a way that you deliver it to the target tissue and the cells have the desired uptake, which is not trivial to say the least. Many things can go wrong: your PROTAC can have bad ADMET properties, and I assume it is also hard to know in advance whether it has a significant off-target effect, i.e. it degrades other substrate proteins as well.
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Hi All,
I'm doing Y2H screening using Clontech system. The problem is, along with my positive, the negative controls also grown. While all the medium, components and procedure is as per protocol. Please suggest possible reasons and solutions.
Thanks
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Hi i am Dr. Asif Ali, if you want to know about positive and negative control please go though this link.
It will guide you a detailed protocol
Regards
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Hello,
I am conducting a sandwich-type ELISA. It is to see if small molecules can interfere with a protein-protein interaction. As this interaction would occur in the human body, may I heat my ELISA at 37C for an incubation of 2-3 hours? Will this have detrimental effects on my ELISA such as releasing the capture antibody from the plate or disrupting interactions from previous coating steps?
Thank you!
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In general ELISA requires to incubate at 37 degree up to 1 to 2 hrs for secondary HRP antibody to bind to antigen. For some special ELISA requires antigen overnight incubation at 37 degree, like in my case HMGB1-ELISA require to incubate at 37 degrees for 20 to 24 hrs. So, I would think in your case ELISA should be okay to incubate 2-3 hrs at 37 degree.
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The idea is to measure the hydro- protein sizes in various solvents (eg. with high ionic strength, EtOH/Water, other organic/water solvents), then, due to the extremely low solubility of some organic solvents in water, in some organic solvent/water systems, the final samples are not "clean". It would be a problem, right? Ethanol is miscible with water, then, it is ok?
Appreciate for any comments, thanks.
Best,
Yuhong
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it is right. The solvents shoud be complete dissolved and "clean". Furthermore, for solvent mixtures you have to measure or calculate the viscosity. The viscosity plays an important role for the hydrodynamic radius by the Stokes-Einstein equation. I would recommand you to clean the cuvette and your solvents. Due to large dust particles or other impurities it could be a problem to see you particles. Bigger particles scatters the light much more in the ratio diameter^6. So, a 100 nm scatters the light 1.000.000times more than a 10 nm particles.
Greetings,
Maximilian
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Is this the same as 4 angstrom which is the distance needed to make contact? And, also is chimera a reliable software to find contacts between different chains of a protein?
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Dear all,
I want to analyse my protein-protein docking complex using DIMPLOT. However i got an error message
LIGPLOT failed-No atom coords found
Possibly this case too difficult to plot.
May i know, how I'm going to solve this error messsage.
I really need a help.
thank you.
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I have noticed that this happens when you have a docking with very large molecules. For example, when you make a molecular docking between a protein and a peptide of 10 or more amino acids. So I cut the molecule in two, save their respective pdb files, and analyze separately with ligplot. I leave you the steps in the document.
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I am going to check effect of various SNP of different genes on disease susceptibility. For this, I need to use Multifactor Dimensionality Reduction (MDR) software to analyze gene-gene interaction. I have never used this software before, so can anyone share their experience about how to use this software. 
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I used to work on gene polymorphism, but I no longer work in this field. I used this software once, but at that time I took help from my lab mate. So, I do not remember how I used this software.
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As we know STRING is a very good tool for the Protein-Protein interaction analysis. But we can analyze only 2000 protein. if it is more than 2000, is there any tool for PPI analysis.
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There are many tools/Databases for PPI network analysis. For example, SIGNOR, Regnetworks, APID, HAMDB etc.
In all these networks, you can construct a PPI network based on attributes like - disease, cellular localization etc.
Some basic analysis can also be performed in Cytoscape.
You can check the following articles for more info
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Dear All
We have revealed good results of certain fungal against cercaria of schistsoma. No we are seeking a good online tool, or any other methods to predict the extent of interaction between this chemical and proteins in the schistsoma.
Any help ?
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Assume that, I am using magnetic bead that is modified with CD9 antibody and captured the exosome. Now I want to collect only specifically captured exosome leaving the magnetic bead/antibody complex.
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To be honest it is challenging, I know and have tried the protocol using low pH (2.8) glycine buffer to break the Ab-exosomal membrane protein association.And then neutralize with high pH.9 Tria buffer. But it is difficult to check the eluted exosomes' bioactivity. as well as the elution efficiency.(I checked TEM image there are vesicled eluted but not sure to what.extent they are damaged using the acidic condition)
Maybe you can do co-culture with control setting to test if the eluted vesicles are still working or not?
A better idea I would suggest is to design a responsive chemcial ligand on the beads for vesicle capture-release and this need help with surface chemistry design.
Good luck
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Hi I am trying to express my protein R in tobacco leaves and do a CoIP, but I can't detect the protein R in CoIP input.
I took the same tissue and added 8M Urea plus LDS protein buffer, protein R shows as a distinct band at the correct size. But when I grind the tissue and put in CoIP lysis buffer, after centrifugation, I put the input in LDS, I couldn't detect my protein, I only see a smear.
Does anyone know what went wrong?
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ok, we´re working with the Plant Protease Inhibitor cocktail from Sigma (P9599).
This works also fine.
Best and good luck furthermore!
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I am trying to express and isolate a protein in E. coli. The protein is supposed to have high prevalence of non-polar residues. Would that create problems in isolating the protein using a His tag column? If yes, are there other ways we can isolate proteins which are rich in non-polar residues?
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High prevalence of non-polar residue in protein (his-tagged) is not going to affect its purification by affinity chromatography. Membrane proteins have a high percentage of non-polar amino acids. You can purify the protein under non-denaturing conditions & run on an SDS-PAGE to check the presence of desired protein. If your protein is a membrane one, do not boil your sample before SDS-PAGE as it can form aggregates. Heat it to 70c for 15mins.
If the protein is not soluble, then you have to purify the protein under denaturing conditions & refold it.
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Hello everybody,
I work on plant science, more specificlly in plant pathogen interaction. In my project I need to analyse an interaction of some proteins related to host and virus interaction, but before I'd like to do this analyses in silico.
Do you have any idea of a good software/program, online or not?
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Dear Athithan,
you can use PDBsum. I think it is very useful and intuitive.
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Could anyone tell me an alternative to Ligplot+ software which could be used to determine the amino acids and the types of bonding between the interacting amino acids in protein protein interaction analysis?
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@ Bipasha Bhattacharjee you can submit your docked complex to PDBsum under Generate option available in the homepage menu of PDBsum. It will give you ligpot interactions with diagramatic representation for docked complex as well as the residuewise interaction listed with bond distances in the interaction file. Hope this will solve your problem.
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in which i get to know about active site.
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I am using the VMD plugin QwikMD to do MD simulations of my protein-protein complexes. Hereafter I am using the NAMD2 analysis to calculate my total energy of the system along with all of the other energies. I want to find out whether my reasoning is correct: If I have protein A, B and the complex AB, would it be correct to take the interaction energy as the average total energy of each separate simulation and have Binding energy = energyAB - energyA - energyB? I am also doing a simulation in the presence of metal and without metal where binding energy would be BE = MetalBound - MetalFree. Is this the correct approach for a simple estimate of binding energy?
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You can use MolAICal ( https://molaical.github.io) to calculate the MM/GBSA based on the simulated results of NAMD2. Please see the tutorial: MM/GBSA tutorial by NAMD and MolAICal in the website: https://molaical.github.io/tutorial.html
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As I checked some papers like the following one, the authors consider an amino acid as hot-spot if mutation to the alanine change the DeltaDeltaG > 2 kcal/mol.
My question is what about DeltaDeltaG < -2 kcal/mol? If a mutation change the DeltaDeltaG less than -2 we can consider that amino acid as hot spot?
Thank you,
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The hotspot of a protein can be defined as the single most amino acid responsible for binding of incoming molecule. This is newly developed concept coming out of CARd analysis from carbon value. According to CARd one, amino acids mostly aromatic one are responsible for the binding and also available for effective locking by adjoining portion by forming internal COD. Effective mean carbon distributed according to principle of cohesiveness of arising from carbon score of 31.44 percent.
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Suppose I am given the amino acid sequence of two proteins in Fasta format. Seeing the sequences, how will I predict whether these 2 proteins will interact or not?
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From sequence information one can identify active regions and all, but obsolute affinity test between proteins were to be derived from 3D structures. CARd analysis can be used for active regions identification. All available information for specific one can be utilised for association acceptance. Accordingly carbon value of binding sites are to be given importance for validation which is not done so far as for as I know, available information regarding association principle are adequate enough but not at all understood clearly, carbon point of view needed to be considered here. I hope it start working in the near future. For now understanding is important, availability think later on.
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I want to find the residues of human ACE 2 which interact with the spike protein of Covid-19?
or can anyone tell me a method to find the interaction residue for this PDB file?
PBD ID: 6lzg
thank you.
Be safe form covid-19
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by downloading the protein and opening it with Biovia Discovery studio I checked the mentioned active site residues and here are they:
HIS374 HIS 378 GLU 402 OR (GLN340 GLU57 THR55 ASN53) OR (LYS26 ASN90) or (ASN322 MET323) or (PHE338 PHE342 PHE343).
you also can check the actual active site residues from Uniprot.
some researchers consider the native ligands that comes with the crystal structure position is the active site.
or you can use online servers for predicting it for you (which I don't prefer) such as:
best regards
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What is the suitability and safety of SCP for human consumption?
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Please take a look at this useful RG link.
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Transform e.coli Bl21 DE3 with IPTG inducible pET. I grow the bacteria in 15ml of LB medium, with 0.2% D-glucose, and 15 microliters of ampicillin, then I add it to 100 of LB medium with 0.2% D-glucose and 100 microliters of ampicillin, I wait 4 hours , and I induce with IPTG 0.5mM 100μl , I wait 4 hours, and I centrifuge, the pellet I treat with Urea 6M 500μl, lysozyme 50μl, Protease inhibitors 10x 50μl, EDTA 2% 50μl, then I leave it overnight, the next day I do thermal shock 10 min at -80 ° C and 15 min thaw, that 5 times. then centrifuge and I do W.blot of my supernadant, I use buffer laemlii for ride the protein in the gel.
My protein of interest is not expressed or do I get good bands on the PVDF membrane or do I use better nitrocellulose?
What recommendations would you give me?
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Suggest looking at kinetics of induction, not just a single 4 hour time point. Remove samples periodically for analysis, we just use SDS-PAGE of the samples, maybe one, two, and three hours after induction. We have a protein that exhibits maximal expression at ~1 hr in, then rapidly decreases in levels.
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What are various options available while determining the binding affinity of a protein to it's target? In my case the target is also a protein. Are there any standard physical methods to determine the binding affinity?
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There are many methods. Biophysical methods are best applied to well-purified proteins, preferably available in multi-mg quantity. Here are some methods.
  • Equilibrium dialysis
  • Intrinsic (mainly tryptophan) fluorescence
  • 2-D protein-observe NMR
  • microscale thermophoresis
  • differential scanning fluorescence
  • isothermal titration calorimetry
  • surface plasmon resonance
  • the Hummel-Dreyer method
  • equilibrium ultrafiltration
  • displacement of a fluorescent ligand, measured by fluorescence intensity or polarization changes
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I am trying to do a kinetics characterisation of AB-40 and AB-42 (they are recombinant peptides produced in the laboratory) but it seems that the samples ar alredy aggregated when I try to do the kinetics mesurement. This peptids, after the purification procedure, are storaged at -20°C and in a solution with a pH of at least 8. I was trying to disaggregate using sonication but it doen't work so well, i was wondering if there is another method I can use for the sample preparation.
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You refer to amyloid beta peptides that are highly hydrophobic peptides. I came across the following paper that might be useful to you:
Broersen, K., Jonckheere, W., Rozenski, J., Vandersteen, A., Pauwels, K., Pastore, A., ... & Schymkowitz, J. (2011). A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer's disease. Protein Engineering, Design & Selection, 24(9), 743-750.
If you have a powder form of the peptide you could ‘simply’ use DMSO or TFE (after TFA treatment) see for example:
Killian, J. A., Keller, R. C. A., Struyve, M., De Kroon, A. I. P. M., Tommassen, J., & De Kruijff, B. (1990). Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes. Biochemistry, 29(35), 8131-8137.
Where a highly hydrophobic signal peptide was used.
Hope this helps you a bit further.
Best regards.
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I performed a differential expression analysis and I have a list of DE human genes. now, I want to visualize DEGs by Cytoscape (showing up-regulated and down-regulated proteins with different shapes and set the size of the nods on the base of levels of expression.) but this work needs a network and expression data.how should I obtain these files? And after that how to connect them to build a network?
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Hello Momeni, I got what you want, you follow this questions and answers. It will help you.
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Hi, I have to see some protein-protein interaction for which I am using the yeast two hybrid system. After constructing the fusion GBD and GAD constructs (in pGBD C1 and pGAD C1 vectors) I want to verify the expression by western blotting using anti GBD and anti GAD antibodies.So, my question is -can any body tell me a source of antibodies against the Gal binding domain(GAD) and Gal activation domain (GBD) that I can use for the above mentioned purpose?
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How do we use Y2H in our research work? Plz guid me..
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Our truncated protein expressions (labeled by the HA tag) in lane 4 and 5 were supposed to be matched with the mRNA expressions in lane of 293FT-4 and 293FT-5. However, the different truncated proteins seems to be equal of molecular weight. We changed different vendors of HA antibody but the results were the same. And we are sure our truncated protein expression sequences are correctly with Kozak sequence followed by the initiation codon and stop condon, and the CDS length is the integer folds of 3, thus it seems to be an error of protein translation.
Any suggestions? Thanks a lot~!
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Hi again,
If the host for expression is eucaryotic then polyA is definitely essential... About the WB: did you run a negative control sample issued from a cell transfected with an empty plasmid? It is just to make sure the band is specific to the presence of your gene...
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Hi,
I am trying to express 2 seperated proteins say Protein A (200kd) and protein B (100kd) in E.coli.
I need to express them from 2 seperate vector but want them to fuse once they are translated in the cell.
Is there any fusion domain anyone has used that I can use.
Thanks in advance
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what you are looking for are inteins (which can basically work as a protein ligases) see the attached review. There are some modified forms of them that catalyze the reaction when a small molecule is added (perhaps this is what you need).
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Dear All,
In order to understand the protein interactome of my gene of interest, I am planning to tag the endogenous locus of the gene with V5 epitope and to perform mass spectrometry-based proteomic analysis, followed by co-immunoprecipitation against V5 epitope.
Do you think that V5 epitope is much suitable for this approach?
What are methodologies that I can use to reduce the background and to increase the resolution of my results?
Will it be better to have a crosslinking step/ method for the procedure?
Thank you with warm regards
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Hi Chamara,
It's worth to compare 3X-FLAG peptide and enterokinase digestion in elution efficiency. You'll probably want to remove enterokinase after cleavage for your downstream experiments. One way to do that is to incubate your elutions with anti-enterokinase resin and collect the flowthrough.
With peptide competion, you can remove excess free 3X-FLAG peptide from your eluted complex with a spin concentrator at low molecular weight cutoff, dialysis membrane, or SDS-PAGE.
Thanks,
Barry
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I want to incorporate two proteins (6 and 2 TMDs) as a complex into nanodiscs. 
I am wondering if it would be better to express and co-purify them together (either from a single plasmid or with two different ones). I tagged them differently (C-term with His/Strep) to be sure I have both in my discs and tried to express them from pETDUET1 using both MCSs (0.1mM IPTG; diff. temperatures & diff. strains C41/C43/pLysE/Rosetta).
In this case only the His-Tag version in the 2MCS is there but expressing them separately works – more or less – fine, no matter if I use the 1st or 2nd MCS or His/Strep tag. Should I enhance IPTG level or clone both into a single MCS (as a kind of operon or even a fusion protein)?
The alternative would be to purify them in parallel and combine them during the nanodisc step. But in that case, I assume the detergent may inhibit complex formation.
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If there proteins interact with each other and form a complex I think of no reason it won't work. There are examples of complex reconstitution into NDs. See for example SecYEG a heterotrimeric complex.
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Hello
I am doing western blot for human T-cells with CD28 antibody and I detect bands at around 88kDa as dimers which is as what the companies say. The question is that the sequence of CD28 contains 202 amino acids as total. The average molecular weight of single amino acid is 110 dalton so when I make the calculation I get 22.22 Kda for the monomer multiplied by 2 for dimer so 44.44.
The companies (such as the provided link) say it is 44 for monomer and 88 for dimer. Am I missing something or my calculation is wrong?
Thanks
Salem
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CD28 is a glycoprotein, so maybe the extra 22 kDa per monomer is carbohydrate.
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I wish to perform biotinylated protein interaction pull down assay. please share your expertise and experience on this. Also the thermo fisher protocol suggests to work with 50 ug/100ul of protein. Has anyone worked with other protein concentrations? Please suggest. Thank you
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What method for purifying biotinylated proteins will you utilize? I used streptavidin magnetic beads because it's easy to do a lot of samples at one time and there's a very effective protocol for eluting the proteins.
Cheah, J. S., & Yamada, S. (2017). A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat. Biochemical and biophysical research communications, 493(4), 1522-1527.
With this protocol, I used 300 ug protein in 100 uL, but I don't see why you can't use a larger volume and an even more concentrated solution. If you plan to identify the proteins by MS, then you should consider using about 1 mg of protein, otherwise, you will not see low abundant proteins. Also, carboxylases naturally contain a biotin group so you will not be able to determine if your protein interacts with those using this assay.
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Dear all,
I am trying to check the interaction between two of my proteins of interest. However before trying any biophysical technique such as SPR/ITC/MST etc, I used normal superdex-200 gel filtration chromatography. I used protein A (Receptor; ~40 kDa) and protein B (Ligand; ~31kDa) in purified form and mixed together with 1:2 molar ratio at temp. 25 degree for 1 hour. Now when I performed Superdex-200 with three individual runs (Ligand, Receptor and Complex). each time I get a surprising result wherein the complex has more retention volume than the Receptor it self (attached result brown: complex, cyan: receptor and blue: ligand).
When I run the peak fractions from each run through SDS PAGE, I get the two different protein bands in the complex run (attached result; lane 1-3 is the Ligand and Lane 4-6 Receptor and lane7-9 Complex peak fractions). Please suggest what could be the possibility of such result. The complex formation shows about 1 degree (57 to 58 degree C) rise in melting temperature every time as compare to their individual ones. Buffer used for the protein are same 1X TBST pH=7.6.
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There is not much difference in elution time between the three samples. The small differences may not be significant. Although your expectation is that the complex would elute earlier than the individual proteins, this might not happen for a variety of reasons: (1) No stable complex was formed, (2) the complex formed, but it eluted at about the same time as the individual components because of a more compact shape of the complex compared to the individual components, (3) at least one of the component proteins interacts with the column resin strongly enough that this interaction dominates the elution profile of the complex so that it elutes at the same time as that component.
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I am working on a human helicase protein and when I load them on a SEC column Superdex 200 (GE), I don't get a good recovery. In addition, when I load my protein-DNA complex into the same column the complex does not come out at all. Had anyone experienced a similar problem?
Also, had anyone here tried to purify a protein-Holliday junction complex? I have a mixture of two oligomers (homo) in my protein, and one of the oligomer binds to HJ, while the other does not. For further studies I would like to purify the protein-HJ complex from the non-binding oligomer of the protein, but not finding a good way. Please share your thoughts/experiences.
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hi, i'm experiencing something similar. there is a protein portion that elute during a log range of volumes and at the BN-PAGE analysis it looks quite heavy. i don't understand why it elute along all the volumes from the aggregates range to the monomers range. i was thinking the same, that my protein being highly glycosilated stick to the column. dos it make any sense? any suggestion?
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I have a bacterial protein 'X' and I want to check if it interacts with any host protein (Human/Mouse). Is there any tool available that can predict interspecies interactome, like how STRING analyses protein-protein interactions within a species?
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Hi there,
I don't think there is a good bioinformatic tool for this. But you could express the bacterial protein with a GST-tag and purify it.
Afterwards, you can prepare eukaryotic tissue lysates and check if you can pull down interaction partners.
Good luck,
Sebastian
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Now, I'm trying to finding some software that can serve me to analyze:
- protein - protein interaction
- drug (known structure) - protein interaction
- drug - drug interaction
Can you recommended a free software for this?
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Try this site gives a list of a variety. you have to sign up for a trial and you can access a lot of tools for any drug specificity tool you like.
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Hi everyone. I want to build a PPI network using STRING (directly or through cytoscape) out of a list of differentially expressed genes, however, I want to separate the network in up and downregulated genes but I can`t find a way to do that. In ClueGo for example I can create different clusters before building the network
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The cytoscape platform, if you've downloaded and installed the application, would allow you to do that. In short, you would
1) import a network (from NDex or file or list) if you use NDex you can look for a network with "STRING" and whatever your biological system is. If you have a small list you can also copy and paste your accession list.
2) import a table from file,. This would be your excel sheet with accession with fold change, pvalues etc. Essentially your experiment
3) In the style tab you would then go to label, and select the column containing your proteins/genes. Select mapping type as continuous (this allows you to make downregulated blue and upregulated red for eg)
4) GO to the select tab: Column Filter and select your fold-change column and set your cut-off e.g. log2 (1) for a 2-fold change. So it will only show proteins/genes that are upregulated by 2-fold or more.
5) You can use 'apps' if you wanted to generate networks based on GO annotations as well.
The manual http://manual.cytoscape.org/en/3.7.1/ describes it in better detail.
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I am trying to pulldown a protein of 300 kDa using protein G agarose beads. I used two negative controls, one pulldown with just beads (without primary antibody) and another pulldown with another non-specific primary antibody. I used rabbit primary antibody for pulldown and mouse primary antibody for detection by Western blot. A band at 300 kDa was detected in pulldown sample with specific antibody, total cell lysate and pulldown sample with only beads. However, when I probed for another protein of 43 kDa (that might be interacting with the 300 kDa protein), a band at 54 kD (band density more than that of 300 kDa protein) was seen in the pulldown sample with non-specific primary antibody, total cell lysate, pulldown sample with specific antibody and pulldown sample with only beads. Is it due to the nonspecific binding of the protein (54 kDa) to the beads or the primary antibody is interacting nonspecifically to the unknown protein of 54 kDa? If it is due to the nonspecificity then how can I avoid it.
Here is the work flow of my Co-IP experiment:
1. Pre-clearing of lysate: 500 ug of cell lysate was mixed with 40 ul of 50% bead slurry and incubated for 1h at 4° C. Later, the mixture was centrifuged and the supernatant was collected for pulldown.
2. Antibody was added to the collected supernatant and overnight incubated at 4° C.
3. 40ul of 50% bead slurry was added to the antibody-protein mixture and further incubated for 1h at 4° C.
4. The beads were washed three times and 20ul of 2x laemmli buffer was added and heated for 10 minutes at 100° C. Western blot was done.
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@Ian R Wilkinson,
Thank you for your valuable suggestions. I take them into consideration in my next IP experiment.
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I want to study possible protein-protein interaction using iso thermal calorimetry, can anyone provide any methodology, protocol paper for the same.
Thanks in advance.
timir
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mbSUS has been used successfully by several researchers to screen for protein-protein interaction for membrane proteins but I could not find if anyone has used it to screen cDNA library. if it is possible, can anyone suggest how to go about generating the library using the same system?
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Did u find out?
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Hi all,
I recently performed BiFC experiment to confirm the direct protein protein interaction between two protein. However, I also tried to validate specific interaction through western blot with the antibody against FL-GFP for the cells transfected with either vc155-protein1 or VN173-protein2. I observed positve GFP signal in Western blot. is it expected? I have been following the protocol of "
Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo
Hsien-Tsung Lai1 and Cheng-Ming Chiang2,* "
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Hi there,
The idea of BiFC is to express halves of GFP fused to your proteins of interest. If the POIs interact, you could bring both halves together to get a functional GFP.
So if there is one half of a GFP expressed in the cell, I am not surprised that a GFP antibody can detect it. The molecular weight of the fusipn protein should be a bit smaller than the MW of your protein fused to GFP. However, unless you have a small POI and run a GFP-fusion protein on the same gel, you might not be able to see the difference in MW.
Best,
Sebastian
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Hi,
I need to check unknown proten-protein interaction by Gel shift assay, but i dont know the interacting partners. So, my questions are as follows:
1. What kind of transfer buffer and membrane to use for transferring the bands from native PAGE ? Also, what should be the ideal transfer time/energy/current?
2. Can I use primary antibodies specific for confocal/FACS/Flow cytometry/IP application or I need to use conformational antigen specific antobodies? If i can use the confocal/FACS/IP antibodies, shall I use polyclonal, instead of monoclonal antibodies?
Any literature suggestion will also be fine.
Thanks.
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Western blotting is usually performed with proteins denatured with SDS, which gives them a high negative charge (about 1 molecule of SDS bound per 3 amino acids). If you perform native PAGE, SDS is not present and the protein charge much lower. Blotting will be more difficult, and the protocol will have to be adapted to the specific case.
  • The gel will have to be equillibrated with transfer buffer for long enough that the intended ionic conditions are met throughout the gel, but not so long that diffusional band spreading occurs. I'd try 1 h first. Including SDS in the transfer buffer may be a good idea to ease the protein out of the gel. Then I'd incubate at elevated temperature (say, 40 °C) to speed detergent binding to the protein. If you want to make zymograms (use enzymatic activity for staining) detergent of course is not an option.
  • Composition of the transfer buffer: Towbin's transfer buffer after SDS-PAGE is in effect electrophoresis buffer with added methanol. Methanol increases small protein binding to the membrane, but prevents mobilisation of large proteins from the gel. The SDS present has, in effect, the opposite effect. doi:10.1006/abio.1993.1313 describes a semi-dry system with MeOH on the membrane and SDS on the gel side. doi:10.1016/0003-2697(86)90207-1 describes a very simple transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, with 50 µM SDS added for large proteins) that in my hands works better than Towbin's. I'd try either that or the electrophoresis buffer used (which may have to be diluted to reduce current - and hence heat - during blotting).
  • Membrane: PVDF is more expensive than NC, but has a lot of advantages. Its protein binding capacity and affinity are much higher, protein slipping through the membrane is not an issue. It is mechanically stronger and will not fragment during the later incubation steps. And, if protein sequencing is intended, the cut-out band on PVDF can be used directly in gas-phase sequencers.
  • Blotting direction: In SDS-PAGE all proteins are negatively charged because the charge of the bound SDS exceeds by far the charge of any amino acids in the protein. Hence, the membrane has to be on the positive side during blotting. In native electrophoresis protein net charge is determined by pI and buffer pH, and some proteins may move to the positive side, others to the negative. Hence, you need membranes on both sides of the gel. For the same reason, you need to add the protein sample to the middle of the gel initially, this requires horizontal electrophoresis.
  • Blotting time and conditions will have to be optimised. You want to shoot for about 30 V, buffer concentration should be low enough that the current doesn't exceed 200 mA for a minigel. In my hands, tank blotters work better than semi-dry systems and are easier to cool. However, if you want different buffers for membrane and gel as discussed above, you are limited to semi-dry.
  • Antibodies: Western-blotting after SDS-PAGE requires sequence-specific antibodies, because protein conformation is destroyed. After native electrophoresis, conformational antibodies are worth a try, but it would be a good idea to also use a sequence-specific antibody for control (how native is native electrophoresis, anyway ;-) ).
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We would like to scan protein/peptide slides. Mainly, these are microscopy-sized slides with proteins dotted onto them, and then we visualize binding of another protein to these dots, using fluorescent antibodies.
The problem is that we only have the basic model of the Typhoon Trio available, which doesn't come with a slide holder. This slide holder is needed so that your mounted slides do not touch the surface of the scanner, which would cause smearing of the dots.
Do you have suggestions how we could mount and scan the slides without smearing? Any DIY solutions out there?
Thanks in advance.
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Did you come up with a solution to this? I am facing same problem
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We are testing the complex formations between soluble proteins and different organelles by co-IP experiments using the protein of interest and compartmental markers as prey and bait, respectively. Some of our results were positive. And, we want to further understand whether changes in lipid composition mediates the association.
The best scenario would be that cellular membranes were disrupted while protein of interest associating with the membrane microdomains in specific organelles. If so, we'd like to change the membrane composition with exogenous lipids and examine their effects on the protein association.
So, I am wondering what is the recommended lysis buffer for this purpose, if it makes sense. Additionally, I have no idea how to validate that patches of cellular membranes are presented in the tissue lysate.
Please let me know if you have tried similar experiments or have any comments/ideas.
Thank you.
P.S. We're using Drosophila head extracts with 5 mM HEPES pH 7.4,
100 mM NaCl, 0.3% Triton X-100, and protease inhibitors. The lysis buffer worked well for the co-IP experiments but, as I mentioned, we don't know if the membranes were still there or it's just protein complexes.
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Thank you for your reply.
I think we will try different concentration of non-ionic detergent first. Nitrogen cavitation seems nice for studying localizations of protein interactions; however, we don't have the tools for that.
And, after a second thought, it sees like using in vitro liposome with recombinant protein to identify the effect of specific lipid compositions on membrane association of protein might be better (e.g. DOI: 10.1126/science.1092571 Fig.2B)
Once again, thank you for your comments.
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I am not sure whether I should use ANS binding or SDS binding.
If I use ANS binding, do I have to detect 5 concentrations for every protein sample in order to get the initial slope?
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I'm adding a tag to a protein with crisprCas9 in order find its interactors and localization. I'm thinking to use HeLa cells but they have a strange karyotype..what type of cells would you use instead?
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Cells that are not cancerous would be preferred. Like hepaRG. It is although not a primary cell but not a cancer cell either. And maintains close smilarities with primary cell
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What methods can be used to study a competitive interactions between two different proteins (eg. A & B) for a common binding motif on protein (C).
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1. Fluorescence resonance energy transfer (FRET). Attach a fluorescent donor label on protein A and acceptor label on the peptide (or vice versa) such that there is FRET between the labels, as measured by fluorescence intensity, when the peptide is bound to A. If B competes with A for peptide, then the FRET signal will decrease as the B concentration increases.
2. Chemical crosslinking. Use a chemical crosslinker that causes covalent bond formation between A and peptide. This could be an exogenous compound, or the peptide could be synthesized with a reactive group. The reaction could be observed by a change in mass of A on SDS-PAGE, if the peptide is large enough. Determine whether adding B results in less crosslinking of A with peptide accompanied by an increase in the mass of B. Intact protein mass spectrometry would be a more exact method of demonstrating this. In either case, it relies on there being a substantial mass difference between A and B.
3. Surface plasmon resonance (SPR). If there is a substantial mass difference between A and B, then SPR could be used to distinguish between A and B binding to immobilized peptide by a change in the size of the resonance signal.
It might not be necessary to do this experiment as a competition between A and B. It might be enough just to measure the Kds for the peptide-A and peptide-B interactions separately. This could be done by a number of different methods, including isothermal titration calorimetry (ITC), differential scanning fluorescence, FRET, fluorescence polarization, equilibrium dialysis, SPR, microscale thermophoresis, intrinsic protein fluorescence, etc.
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Hi Fellow Research Folks,
I have been looking for a transgenic mouse model to track the temporal association of two known protein interaction. I came across a paper "Noninvasive imaging of protein–protein interactions in living animals' by Luker et al. 2002. But I was wondering anyone has worked on a similar scenario and can shed some more light about any existing tool to look at such interactions without sacrificing the mice(Imaging modalities like Xray, MRI,CT or PET etc).
Thanks a lot!!
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Nisha Kuzhuppilly Ramakrishnan : Thanks a lot. I was leaning towards that idea as well but thanks for the heads-up!!
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Suppose 2 genes produce 2 proteins which would be binding. After the genes are turned on and the proteins are produced, then genes can be turned off and the proteins produced can bind with each other even after the genes were turned off right?
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Many weak bonds are needed to enable a protein to bind tightly to a second molecule, which is called a ligand for the protein. ... The region of a protein that associates with a ligand, known as the ligand's binding site, usually consists of a cavity in the protein surface formed by a particular arrangement of amino acids.
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Without use of fancy equipment like ITC
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A more powerful, new generation of the Y2H has been developed that gives you much more precise hits (no false positives) and more hits from 10-100x more comprehensible data than the old conventional Y2H. Its called the NGIS. It saves you lots of time and cost down the road. Not only screens for novel interactions, but can also map the binding sites. Check out www.nextinteractions.com techology tab. Testimonial tab: https://nextinteractions.com/discover-crucial-protein-protein-interactions/
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I am planing BiFC assays and before I want to start I have some general questions:
What is the strongest BiFC system? At the moment I could not say anything about the strength of my protein-protein interaction. It is an interaction between a membrane protein and a cytoplasmic protein.
Does anyone have experience with the BiFC assays in the zebrafish?
Is it possible to screen a library with BiFC for novel interaction partners?
What is the best linker?
Thanks for answering my questions!
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why certain FP tags are good for BIFC, for eg. the moment you use UV light or laser too detect signal in your samples. The UV light stress activates ROS- reactive oxygen species which makes the FPs to get photobleached hence try use Stable FPs . In my experiment CyanFP has the fastest photobleaching
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I want to know if there is an online server for ligand-protein complex docking with another protein. The available renowned protein-protein docking servers like ClusPro, HADDOCK, PyDock etc. either remove or give an error when I try to upload the ligand-protein complex as the receptor pdb file. How can I verify that binding of a ligand to a receptor induces a conformational change or directly blocks the interacting surface in which the native proteins couldn’t bind effectively thereafter?
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Ardavan Abiri If i understand correctly, you have a protein-A with bound ligand and you wish to dock another protein-B to this ligand-protein complex.
Well its true that protein-protein docking softwares do not handle ligand well.
Now i am presuming you have two conformations of protein-A :- (1) its native state and (2) protein-A bound to the ligand which has undergone a conformational change upon binding.
In such cases you can first dock protein-A (native state) with protein-B and predict the interface. Then you can dock the alternative conformation of protein-A with protein-B.
You can then compare the top predicted interfaces between the two types of docking experiments. If the ligand disrupts the binding, then this comparison of docking experiments might provide an indicator.
hope this helps.
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Hi!
I want to generate a list of putative binding partners for my protein of interest by essentially BLASTing for the ligand which binds to EVH1 domains, FPxΦP. Is there a database where I could enter this, and it would search for all of the possible combinations of my motif? It seems that there are a ton of options out there, so I'm a little overwhelmed with trying to figure out which direction to go! Thanks in advance!
Sara
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Hi Sara,
So you want to find a set of putative ligands and you already know at least one ligand, right? You can use blast to search for motifs with similar sequences, as you said. Additionally you can use CATH (http://www.cathdb.info) or SCOP (http://scop.berkeley.edu) to search for motifs with similar structure. Also string (https://string-db.org/cgi/input.pl) may help you finding putative ligands of the protein you are interested in. Good luck!
ML
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