Guys who's done immunoprecipitation- help me! - (Dec/01/2005 )
I'm trying to show protein-protein interaction using a co-IP approach, starting with pulldown of a protein that is transiently transfected. I know there's not going to be a lot of this protein in the cell (I can't detect it by western blot of transfected cell lysate).
How much cell lysate should I load? Some protocols recommend 500ug. Others suggest to dilute the lysate down to 1ug/mL first. What's the rationale for all this? If the protein of interest is lowly expressed I should load more lysates right? Would too much lysate lead to high non specific protein binding, or would the excess proteins hinder approach of antibody? So if I am using a 1.5mL tube for the IP, what volume or amount of protein would you recommend, for a start? Thanks.
If you can't detect the protein by western, I'd start by troubleshooting that problem first.
In particular, adding sufficient protease inhibitors to ensure my protein is not being degraded
during extraction.
I'd also verify the IP capability of the antibody, probably by taking some purified protein and
using that to verify that the protein can be pulled down with the antibody.
I'm trying to show protein-protein interaction using a co-IP approach, starting with pulldown of a protein that is transiently transfected. I know there's not going to be a lot of this protein in the cell (I can't detect it by western blot of transfected cell lysate).
If you can't detect the protein by western, I'd start by troubleshooting that problem first.
In particular, adding sufficient protease inhibitors to ensure my protein is not being degraded
during extraction.
I'd also verify the IP capability of the antibody, probably by taking some purified protein and
using that to verify that the protein can be pulled down with the antibody.
Hi. Thanks for your response. Yes I did use protease inhibitors, and will attempt to review that aspect. But for western, is the antibody only recognising the primary sequence, or does it need the protein to be properly folded before it binds? After denaturing SDS-PAGE the protein would be denatured, but I am wondering how much renaturing occurs while incubated with antibody.
As for purified protein, unfortunately I'm working with a HA-tagged transmembrane receptor, and its very hard to purify a transmembrane protein. But if I have some other purified HA-tagged protein I could.
I'm trying to show protein-protein interaction using a co-IP approach, starting with pulldown of a protein that is transiently transfected. I know there's not going to be a lot of this protein in the cell (I can't detect it by western blot of transfected cell lysate).
If you can't detect the protein by western, I'd start by troubleshooting that problem first.
In particular, adding sufficient protease inhibitors to ensure my protein is not being degraded
during extraction.
I'd also verify the IP capability of the antibody, probably by taking some purified protein and
using that to verify that the protein can be pulled down with the antibody.
Hi. Thanks for your response. Yes I did use protease inhibitors, and will attempt to review that aspect. But for western, is the antibody only recognising the primary sequence, or does it need the protein to be properly folded before it binds? After denaturing SDS-PAGE the protein would be denatured, but I am wondering how much renaturing occurs while incubated with antibody.
As for purified protein, unfortunately I'm working with a HA-tagged transmembrane receptor, and its very hard to purify a transmembrane protein. But if I have some other purified HA-tagged protein I could.
generally when you're running SDSPAGE, all your proteins are denaturated. there is no renaturation, and AB generally targets the sequence.
i think there are conformational antibodies, but secondary rather..
How much cell lysate should I load? Some protocols recommend 500ug. Others suggest to dilute the lysate down to 1ug/mL first. What's the rationale for all this? If the protein of interest is lowly expressed I should load more lysates right? Would too much lysate lead to high non specific protein binding, or would the excess proteins hinder approach of antibody? So if I am using a 1.5mL tube for the IP, what volume or amount of protein would you recommend, for a start? Thanks.
Hi, I have some experiences and like to share with you with the IP question. I am working on a rare expressed protein (1600/cell) as you. I will list some information downhere
1: Use as much protein extract as you have. Think IP as a concentration column, if your interest is so less and you better increase the starting pool no matter how dilute it is.
2. The buffer. My protein is predicted a TM domain inside the sequence. Try buffer like this 20mM sodium phosphate, 0.5M NaCl, 1%NP-40 pH7.5 in protein extraction and the following IP process.
I can capture my interaction proteins only under this condition and PBS is not working. I use in-house Ab and commercial anti-myc monoclonal Ab, they all work well in this buffer. So try to adjust it a little bit if there is anything concerned, but , leave the 1% NP-40, it's the necessary one I think.
3. Western part. How about do a serious increaseing or dilution of your cell extracts before the IP step. Increase the protein amount in SDS-PAGE and keep increase until you can see the western signal. And this protein conc can serve as a limited protein amount for the IP.
4. Usually, proteins are all denatured in SDS-PAGE so that the eptopes are more exposed. That's way sometimes the Ab can do western well but can't do IP. Watch out for the Ab you purchased, they always have a page to tell you what's the limitation of this antibody. One more thing, polyclonal Ab is more suitable than the monoclonal one in IP experiment, because it rcan ecognize more epitopes.
Good luck^^ allie
Hope u have properly designed positive and negative controls for yr western blot.. only then would yr conclusion suffice and if you do get a good result there.. trying different dilutions of yr extract may help. The control should be processed like yr sample for the SDS PAGE so the question on Ab the denatured protein may be solved, though most Ab should bind a protein in both native and denatured forms ( i suppose so!).
Western blot is a very sensitive technique (upto 10 ng at least) hence u need not worry if ur confident yr lysate has more protein than that. If u think ur protein is much more.. forget the background.. but tell me did u get a signal anyway?
G'luck:)
[quote name='GPCR' date='Dec 1 2005, 03:43 PM' post='33089']
I'm trying to show protein-protein interaction using a co-IP approach, starting with pulldown of a protein that is transiently transfected. I know there's not going to be a lot of this protein in the cell (I can't detect it by western blot of transfected cell lysate).
How much cell lysate should I load? Some protocols recommend 500ug. Others suggest to dilute the lysate down to 1ug/mL first. What's the rationale for all this? If the protein of interest is lowly expressed I should load more lysates right? Would too much lysate lead to high non specific protein binding, or would the excess proteins hinder approach of antibody? So if I am using a 1.5mL tube for the IP, what volume or amount of protein would you recommend, for a start? Thanks.