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probing one protein with multiple antibodies - (Jul/21/2008 )

Hello,
I have a FLAG-tagged protein that I would like to probe with a phosphotyrosine antibody. I have been having lots of trouble with my immunoprecipitations so it is very important that I probe my membrane with FLAG to ensure that I have pulled down equal amounts of protein with my IP (I use FLAG to IP my protein). If I probe with FLAG first, can I follow that with the phosphotyrosine antibody and be certain that the FLAG antibody is not hindering the phospho antibody from recognizing my protein? Neither antibody strips off the membrane very well. My immunoprecipitations are not very efficient so it is not possible for me to split up my samples and run duplicate gels. I would appreciate any advice you can offer. Thanks.

-jlolsen-


You certainly can re-probe a membrane but I recommend you start with your least sensitive antibody first. I know the flag antibody is pretty sensitive so I'd start with the pTyr antibody. My logic is that if the flag signal is too strong it may mask the signal you are hoping to get with the phospho antibody. I assume these are going to be close in size and you are looking for the expressed Flag protein and the phosphorylated form of it. I'm wondering why your IPs are so inefficient since the Flag antibody is really good. How long are you incubating the beads/antibody/lysate? Are you using pre-conjugated beads or are you conjugating yourself? If yourself, are you sure you are using the correct protein (A versus G)? I once set up an IP with the monoclonal myc using the protein A beads which don't bind mouse IgG very well at all. Needless to say it was an unsuccessful IP. Also, if the phospho-antibody and flag antibody are from two different species you can probe with one and use sodium azide to destroy the HRP. Then you can probe with the other and not worry about signal from the first antibody interfering with the second. I generally use 1% NaAz overnight at 4degree. Wash very well and then reprobe. You don't even need to reblock. Otherwise it may just be worth your time to set up the IP twice as big as normal and then split the samples.

-rkay447-

QUOTE (rkay447 @ Jul 21 2008, 01:32 PM)
You certainly can re-probe a membrane but I recommend you start with your least sensitive antibody first. I know the flag antibody is pretty sensitive so I'd start with the pTyr antibody. My logic is that if the flag signal is too strong it may mask the signal you are hoping to get with the phospho antibody. I assume these are going to be close in size and you are looking for the expressed Flag protein and the phosphorylated form of it. I'm wondering why your IPs are so inefficient since the Flag antibody is really good. How long are you incubating the beads/antibody/lysate? Are you using pre-conjugated beads or are you conjugating yourself? If yourself, are you sure you are using the correct protein (A versus G)? I once set up an IP with the monoclonal myc using the protein A beads which don't bind mouse IgG very well at all. Needless to say it was an unsuccessful IP. Also, if the phospho-antibody and flag antibody are from two different species you can probe with one and use sodium azide to destroy the HRP. Then you can probe with the other and not worry about signal from the first antibody interfering with the second. I generally use 1% NaAz overnight at 4degree. Wash very well and then reprobe. You don't even need to reblock. Otherwise it may just be worth your time to set up the IP twice as big as normal and then split the samples.


I use A7r5 cells that are very hard to transfect. I only get about a 20% transfection efficiency, so the expression of my FLAG-tagged protein is low to begin with. I incubate my cell lysate with antibody for 2 hours and then add beads and incubate another 2 hours. My IP antibody is generated in rabbit and I use protein A beads so I know that is not the problem. I have been trouble shooting these IPs for about a year now, and I still have not found a reliable protocol that gives good consistent results. I am trying to convince my PI to buy the FLAG IP kit from Sigma in hopes that using all of their reagents plus their protocol will result in better experiments.

-jlolsen-