Anti-FLAG antibody help, Troubleshooting for Sigma anti-FLAG (A8592) Options

[charles_duh-win]

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.

[bob1]

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.

[charles_duh-win]

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.

[MaggieRoara]

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.