FITC labelled primary antibody OK?

Emir Khatipov khatipovNO at NOuchicago.edu
Mon Oct 22 11:26:36 EST 2001


Martijn,
If you were successful with a single-antibody staining for microscopy, you
most probably will succeed with Western. However, you will have to use more
diluted antibody. From your message I understand that you don't want to do
fluorescent detection of your band, and want to use conjugated secondaries
to develop your Western. If so, ask you provider how your primaries were
FITC labelled. Most probably they were labelled by primary amine, i.e., by
N-terminus (and lysines). The provider of your primaries might give you
information on how many FITC molecules are there per molecule of Ab for the
particular batch you have. I believe that if bound to N-terminus, FITC will
not interfere with binding of the secondaries. It would though if there were
lysines in the recognition sequence and those became labelled, too. However,
I think companies make sure to reduce undesired labelling by controlling
reaction conditions.

Emir

"Martijn van Duijn" <m.v.duijn at amolf.nl> wrote in message
news:3BD4261D.40302 at amolf.nl...
> Hi,
>
> I have been using a FITC conjugated antibody against a HA tag in my
> favourite protein for microscopy purposes. However, I am now interested
> in trying a western blot using this antibody.
> Has anyone tried using (FITC)-conjugated primary antibodies? Does the
> conjugate interfere with the recogition of the primary by the secondary
> antibody? I hope I can avoid buying the unlabelled version of the same
> antibody...
>
> Thanks for any advice!
>
> Martijn van Duijn
>





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