I am doing a co-IP to determine if a protein (CAP37) is binding to a receptor. I am using an antibody against the receptor and agarose beads to pull down the receptor, and then doing a WB for CAP37. It has been suggested to me to strip and reprobe the blot for the receptor to make sure that equal amounts of the receptor are being pulled down between different treatments. I don't see this in a lot of papers and I'm trying to find out if this correct. Technically, I would be using a different antibody for the same protein (the receptor) that I used for the pull down. I'm thinking this could be a problem because the original antibody may chew up the receptor that it detects after a certain amount of time since generally antibodies will destroy the antigens they recognize..Also I'm worried it might get stripped away during the stripping process since the antibody that will be stripped away should be attached to it. Does anyone have any suggestions on this? Please help!!
Firstly, antibodies don't chew up targets, all they do is signal to the immune system that they have identified a binding match. You don't have the chewing-up immune components in your IP reaction. Immunoglobulins have no intrinsic protease activity. You should really do a bit of reading about the basics of antibodies, at least on wiki. It will help you understand the experiment you are doing and aid troubleshooting.
You may not need to strip the membrane if you know the size your receptor will appear on the membrane and you do not have any bands already there ( or anywhere close). Just give the membrane a good wash and incubate with the antibody for the receptor.
It is generally accepted that Co-IP has been checked for equal IP of the protein and also you need to show equal expression of the co-IP protein you are looking at across all the conditions you are investigating. You won't always see it published though, but should be written in the methods section or supplemental section. I always ask to see all the controls when I peer-review manuscripts. Just get into a habit of checking everything while you are doing it now instead of waiting for writing up and not having all the required data.
Hello Mandi,
I would agree with the suggestion to control for equal amounts of receptor being IPed - I would argue that this is a routine control for IP's when comparing different conditions, and without this, it would be very hazardous to draw any conclusions on the association of your co-IPed protein. You mention that antibodies destroy the antigen they bind to - I have never heard of this, and quite frankly, do not understand how this could happen. To my knowledge, a purified antibody has no activity (other than high affinity binding) towards the epitope it was raised, so I don't think you will have to worry about this.
One option is to strip the membrane and re-probe for the receptor. Another option, if your co-IPed protein differs in molecular weight from the receptor, is to run a new gel, transfer, and cut the membrane between the two proteins so that you can blot for both receptor and co-IPed protein.
Good luck!
Firstly, antibodies don't chew up targets, all they do is signal to the immune system that they have identified a binding match. You don't have the chewing-up immune components in your IP reaction. Immunoglobulins have no intrinsic protease activity. You should really do a bit of reading about the basics of antibodies, at least on wiki. It will help you understand the experiment you are doing and aid troubleshooting.
You may not need to strip the membrane if you know the size your receptor will appear on the membrane and you do not have any bands already there ( or anywhere close). Just give the membrane a good wash and incubate with the antibody for the receptor.
It is generally accepted that Co-IP has been checked for equal IP of the protein and also you need to show equal expression of the co-IP protein you are looking at across all the conditions you are investigating. You won't always see it published though, but should be written in the methods section or supplemental section. I always ask to see all the controls when I peer-review manuscripts. Just get into a habit of checking everything while you are doing it now instead of waiting for writing up and not having all the required data.
Hi,
I always check in the same blot, you can do the same or If there is any issue like your protein getting masked with heavy or light chain of IgG, then its better you can run a separate gel to check it out. I don't think, stripping is a better option that too in the IP membrane. Gud luck !!!
Amanda- Yes. It is the T cells that destroy the antigens and not the antibodies. I didn't think this through very well, but thank you for getting me to think it through. All of these responses are very helpful!! ! Roland- I really like the tip of cutting the membrane between the two proteins (since they are a different mw.) Thank you everyone!
This is a nice idea as well. I may do this in the future if the proteins are around the same size and I have issues. I think I can just cut between the membrane for this one. Thank you!
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