T. S. Pillay
tpillay at ucsd.edu
Sun Mar 13 16:32:07 EST 1994
Subject: Prestaining Proteins
From: Richard Near, rocket1 at bu.edu
Date: 11 Mar 1994 22:41:31 GMT
In article <2lqs2r$hdl at news.bu.edu> Richard Near, rocket1 at bu.edu writes:
>I am interested in prestaining some proteins to
>serve as markers in sds gels and/or chromatography.
>I realize that this will affect the apparent Mwt, bu
>t, for my crude applications (at least for now), it will
>serve it's purpose. MY Question: does anyone have a
>simple protocol for prelableling proteins that should
>be stable in sds and/or urea.
>rocket1 at bu.edu
Here's the goods: When I was a graduate student at Cambridge University
a few years ago- I used to make my own prestained standards before they
became commercially available (rainbow markers; kaleidoscope and the
rest). The biggest problem with commercially available markers is the
slight batch to batch variation in apparent MW.
You can avoid this by making your own in bulk ( Does this answer the
question of why make your own?)
The protocol is as follows:
You can choose the colour by using Remazol dyes which come in various
colours- you can make your own "rainbow" (sorry Amersham !!! -
disclaimer: "Rainbow" is a trademark of Amersham International etc
I used Remazol Blue(Sigma), but it can be adapted to other remazol dyes.
Coupling buffer: 50 mM Tris/0.38 M glycine pH8.3
Dissolve Remazol Blue in buffer above at 100 mg/ml.
Make up protein in coupling buffer at 40 mg/ml. Mix with dye in 1: 1
Incubate at room Temp for 2 hours and hey presto your protein is
prestained. Can be used directly without purification or you might want
to remove the excess dye by dialysis or gel filtration. Try it and let
me know how it works.
More information about the Proteins