Can FLAG IP, but cannot detect with Western - (Feb/02/2012 )

I have established a stable cell line expressing FLAG tagged protein of interest. Previously, using RRL, my protein of interest has been successfully IP'ed using M2 FLAG antibody (from Sigma), and the IP was confirmed using radio-labelled methionine for translation.

When I FLAG IP'ed my protein of interest, based on the silver stain compared to non-expressing cells, it seems to have worked (I intend to mass-spec). However, when I check the Western blot with FLAG antibody, I was unable to see my protein of interest (or rather, there's a nonspecific band located at the same molecular weight as my protein of interest that is found in both expressing and non-expressing cells).

There is an antibody detecting my protein of interest; it is able to detect it with other tags, just not FLAG. And this antibody does not pick up endogenous levels of my protein.

I have tried blotting just the cell lysates with FLAG antibody, but had no luck either. Prior to establishing my stable cell line, I verified that my cells still contain my transfected plasmid using RT-PCR.

tl'dr All this to say, I seem to have a working FLAG-tagged protein that can be IP'ed but cannot be detected with Western blot. Help?

-CinogenCar-

Are the cells under some form of selection to keep the insert active (i.e. not methylated or otherwise deactivated)?

I have found the flag antibody to be quite specific for the flag protein, have you optimised the conditions for detection on western blots?

How much lysate are you using for the IP?

-bob1-

bob1 on Thu Feb 2 20:55:27 2012 said:

The cells are being selected by hygromycin (does that answer your question?)

As for the western blots, I've tried everything. Changed lysis buffers, transfer conditions, Ab incubation times, etc. And my colleagues have been working on it for a while as well, and nothing seems to work.

Concerning the IP, I've been using 20 000 ug of protein. I'm quite certain this over-saturates the antibodies on the beads, but there was no signal** in the supernatant, input, or in the pulldown. I've mentioned that I tested the FLAG antibody against whole cell lysates as well, but I see similar results.

I just find it odd that it appears to work for IP but not for the western blot. Is there an explanation for this?

(*note*: Saying "no signal" isn't entirely accurate. There is a non-specific band in my non-expressing cells located at approximately the same kDa as where my protein of interest would be located.) However, assuming the non-specific band is my protein of interest, should I expect to see enrichment of the signal in my over-expressing cells?

-CinogenCar-

Are you sure your antibody works on WB? Some antibodies don't recognise reduced proteins (I don't actually know enough about FLAG to know whether that'll be a problem here).

Also, you say you <"no signal" isn't entirely accurate. There is a non-specific band in my non-expressing cells located at approximately the same kDa as where my protein of interest would be located.) However, assuming the non-specific band is my protein of interest, should I expect to see enrichment of the signal in my over-expressing cells?> but how can you assume that the non-specific is your protein of interest, if this is the "non-expressing" cells they should not have FLAG, and that's what you are detecting... or am I misunderstanding you completely?
What is the size of that non-specific band? Could it be the Ab from the pull down?

-almost a doctor-

almost a doctor on Fri Feb 3 10:32:54 2012 said:

According to the manual, the antibody should be able to be used for both IP and Western detection.

The nonspecific band and where my protein of interest should be located are both at ~130 kDa. It is almost as if the FLAG antibody can detect endogenous levels of my protein of interest, but this is a wild assumption with no base.

-CinogenCar-