use of FLAG Ab - Immunofluorescence (May/27/2007 )

Hi
is somebody using anti-FLAG antibody for immunofluorescence?
I am diluting antibody 1:1000 but still signal is too high.
Have you tried higher dilutions?

Maybe I should dilute more the secondary antibody.

Thanks

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FLAG antibody stains non-specifically in tissues esp. brain . Even diluting it doesnt help much.

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Anti-FLAG staining in brain?
But FLAG is a tag epitope, is not expressed in brain
can you please leaborate on that?

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FLAG is an epitope tag. But if you stain a brain section with FLAG antibody you will get positive staining. Both the brain and retina gives you nonspecific staining. Even take for eg. myc antibody, its not something one can use invivo because again it can give quite a bright background.

You can check this paper for more info.

"Evaluation of epitope tags for protein detection after in vivo CNS gene transfer"

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Thank you for the information but actually I am interesting in staining HEK293T cells with FLAG antibody, the cells transfected witha FLAG fusion gene.
Does anybody have any about the best dilution?

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Hi macedo,

how long do you block your cells? And what kind of blocking reagent do you use?
Maybe you should try to extend your blocking time...

I don't have problems using FLAG-antibody with transfected cells in conc. between 1:600 (when I have a mono-FLAG tag) and 1:1000 (triple-FLAG tag). My blocking reagent is 5% goat-serum in PBS with Mg and Ca.
I use the antibody M2 of Sigma.

Hope those information may be of use for you.

Greetings,
Chakchel

P.S.: My 2nd antibodies I usually dilute 1:500. If you think your high background is because of 2nd antibody you maybe should dilute it more (as you mentioned) and wash you cells more often between 1st and 2nd antibody use?

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Hi Chakchel,
I am using secondary antibody anti mouse alexa 594 diluted 1:200 (and not 1 :400 as I thought)
I will dilute more this secondary and perform longer washes
Thanks

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Okay, good luck!
Please let me/us know if it worked!

Chakchel

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Try a range of different dilutions from 1:500 - 1:5000

You will get the right concentration.

Good Luck !!!

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