Anti-FLAG antibody help - Troubleshooting for Sigma anti-FLAG (A8592) (May/21/2009 )

Hi,

I am trying to detect a FLAG-tagged protein isolated from Xenopus oocyte membranes. I have tried two Sigma antibodies (F7425 and A8592) with little success - I got bands of ~40kDa in both control (water-injected) and FLAG-tagged cRNA-injected oocyte preps, but my protein has an apparent Mw of ~53kDa, suggesting non-specific binding. I have found several papers citing use of the M2 anti-FLAG antibody from Sigma (F1804) which I haven't tried (I would prefer not to spend another £200 just yet).

I am blocking with 5% non-fat milk in 0.05% PBS-TWEEN (and incubating the antibody in the same solution) - has anyone found this affects the specificity of the binding? I should also say that I have tried a range of antibody concentrations for the HRP-conjugated anti-FLAG (A8592) (1:1000, 1:10,000 and 1:20,000) but didn't see a band of the expected size. Has anyone had to use higher concentrations of the anti-FLAG antibody to detect their FLAG-tagged protein? (I thought, reading the literature, that it was quite a sensitive system!).

If anyone could recommend an alternative anti-FLAG antibody that has worked for them in Westerns I would be grateful. Has anyone any experience with the Stratagene or Santa Cruz anti-FLAG ("OctA", in the case of Santa Cruz) antibodies?


Thanks in advance of any help available.

N.

Can you detect your protein besides just the flagged stuff? Is the Flag being expressed? - we have had a case where a vector was mutated for the flag so it was being expressed with the wrong sequence which we couldn't pick up by antibody.

I am aware that you aren't using a vector in this situation; just giving you some ideas.

Hi, thanks for the reply.

Unfortunately, an antibody we raised in rabbit against our protein doesn't work, so I have no positive control for my protein. The sequencing of the FLAG mutant plasmid was fine - the tag was exactly where it was supposed to be, immediately downstream of the start codon (the two Sigma anti-FLAG antibodies I have tried say that they are suitable for N-terminal and N-Met-FLAG tagged proteins).

I do have a positive control for the membrane isolation procedure (another, non-tagged protein expressed in Xenopus oocytes and detected with a commerically available antibody), so I know that this appears to work. My FLAG-tagged protein is functional in the oocytes, with uptake of radiolabelled substrate pretty much identical to the wild-type protein. Could this level of expression be just too low for detection using the anti-FLAG antibody?

My next step is to try a higher antibody concentration (1: 100). Although this will likely make the non-specific band problem worse, hopefully I might start to get a signal from the FLAG-tagged protein).

If anyone has had any success with a similar detection strategy (ideally detecting FLAG-tagged protein in an oocyte membrane lysate) and could recommend an antibody (I keep hoping it's just the Sigma antibodies!), I would be immensely grateful. I saw a post in the archives on this site that looked like it might have just the information I am looking for, but the page is mysteriously unavailable... I can't help but think a rival antibody manufacturer had the page pulled.

Thank again,
N.

HI there:)

I know its some time since you posted this. Have you solved the problem. I use the same Ab as you do and my proteins are around the same size.

The Ab is quite a good one. So i dont think its the Ab's fault. You say you got a band on the 40kDa site instead of 53. You should first check if your plasmid is working. Try expressing it in a common cell line in a transient fasion i.e.using lipofectamine. I use lipofectamine 2000.

Can I take a look at your exposures? just wanna see if you have similiar backgrounds as what i do.