Questions related to Protein-Protein Interaction
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!
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.
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?
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!
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.
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.
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
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.
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?
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?
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?
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.
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.
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 ?
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 !
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!
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.
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!)
What would be the best strategy to optimize a certain crosslinker concentration for my protein interaction studies?
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?
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.
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 .
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?
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.
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?
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.
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?
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.
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.
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.
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 ?
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.
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?
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?
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?
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?
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?
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
Be safe form covid-19
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?
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?
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.
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?
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?
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~!
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
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
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.
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?
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
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.
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.
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?
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
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.
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.
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?
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,* "
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.
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.
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.
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.
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?
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?
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).
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!!
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!
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?
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!