>I am currently trying to purify a protein of 5 kDa, but are having
>problems detecting the protein. As it only contains tree tyrosines and
>no tryptophans the A280 is very low. I have tried running 18% SDS-PA
>gels and a 15 to 25% SDS PA gradient-gels, without luck, the protein
>probably diffuses out of the gel during staining.
>So I am looking for other (easy) ways to detect low MW proteins. Any
>ideas would be appreciated!
>I have heard that adding coomassie to the running buffer instead of
>post-staining the gels could be a solution. Anyone got experience with
>that?
>Thanks, Rasmus
Have you tried detecting the peptide bond (absorbance at ~214 nm)? A280
is what folks often try first, but as you found out, it may not always
work due to few aromatic residues. However, all proteins have the peptide
bond. I've used it in detecting peptide fragments from the digestion
of a 9 kDa protein coming off a reversed phase column. One drawback is
that when you're that far into the UV, background absorbance from other
stuff can be a problem. Blanks will be essential.
Brett
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Brett W. Lennon
Department of Biological Chemistry
The University of Michigan
bwlennon at umich.edu
