labeling carboxylic acids with fluorescein glycine amide
dick at probes.com
Sun Apr 6 21:34:32 EST 1997
It is difficult in general to label carboxylic acids in aqueous
solutions mostly because of the abundance of other nucleophiles,
including amines. The pKa of fluorescein glycine amide's amine is lower
than that of the epsilon amines of lysines so it may be possible to
selectively label carboxylic acids with this reagent.
As a trial I suggest that you activate the protein at the lowest pH it
can tolerate down to about 5 with EDAC (5-10 moles per mole protein?) at
room temp or below for 15 min but must NOT use acetate, citrate or other
carboxylate buffer, which will react with EDAC and you must not use an
amine-containing buffer either (possibly use dilute phosphoric
acid/phosphate buffer). The addition of NHSS
(N-hydroxysulfosuccinimide) was suggested by Staros in Anal Biochem 156,
220 (1986) to improve the coupling yield for similar chemistry and his
paper should be consulted overall. Then add to this 10-29 mole
equivalents of fluorescein glycine amide in a stronger phosphate buffer
to get the pH up to about 7. After some hours remove the excess
reagents by dialysis or gel filtration. If successful, you should see a
fluorescent protein band coming off before the free dye band. You may
have to further adjust reagent ratios and times to get an optimal yield.
If there are free thiols in the protein, you will likely have problems
and there can also be some inter- or intramolecular crosslinking
whenever one uses a carbodiimide for coupling. However, there are
probably no other solutions for labeling carboxylic acids that would be
any better than this. Overall it is much easier to modify thiols or
amines than carboxylic acids in water.
Dick Haugland, President
Molecular Probes, Inc.
dick at probes.com
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