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LUBYPHEL at UTSW.SWMED.EDU LUBYPHEL at UTSW.SWMED.EDU
Mon Jan 9 11:01:15 EST 1995


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Date: Mon, 09 Jan 1995 09:46:15 -0500 (CDT)
From: LUBYPHEL at UTSW.SWMED.EDU
Subject: Re: FITC conjugation of purified proteins
To: RCOVIN at umich.swmed.edu
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Conjugation of FITC to proteins is usually very simple. It binds to 
epsilon-amino groups, i.e. lysines. I have not used the kit, but 
understand it works fine. We label proteins with FITC in 50 mM carbonate-
bicarbonate buffer, pH > 9.0 (the pK of lysines in proteins). FITC is
not very soluble in water so you need to make up a concentrated stock in
anhydrous DMF and dilute it into your protein solution. We usually assume
the reaction will have an efficiency of 10% so we make the final concentration
of FITC 10X M/M with the protein to get one FITC per protein molecule. Also,
the reactive group hydrolyzes off the FITC within about 10 min in aqueous
solutions, so for better labeling we add aliquots of the dye stock solution
to the protein solution every ten minutes for 50 min at room temperature, at
which time we have reached the 10X M/M level, and then stir for 10 mor
minutes. Many people label at 4 degrees C overnight, especially if the
protein is labile. The next problem is to remove free dye that is not
covalently coupled to the protein. Usually, gel filtration on G-25 is 
sufficient to remove most and the rest comes out during dialysis. You
can run an SDS gel and check for free dye in the dye front to see if you
have removed it all. The actual labeling ratio can be determined spectro-
photometrically using the extinction coefficient for FITC at 495 nm and
determining protein by Pierce or BioRad assay. The extinction coefficient
for FITC is very pH dependent and the the sample must be read in a buffer
of pH greater than 8.0. Good luck. KLP

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