gst cross-linking problem

Ralf Weigel rweigel at
Fri May 11 08:27:47 EST 2001

Dear colleagues,

I want to cross-link  a gst-fusion protein to glutathion sepharose for
affinity purification of an antibody. Cross-linking is carried out with
dmp as described in Brew et al., (1975) JBC
250(4):1434-1444. Cross-linking efficiency is tested by analysis of the
resin before (sample A) and after addition of dmp (sample B) using
sds-page. After termination of cross-linking the affinity matrix is mock
eluted with Glycine/HCl pH 2.5 to wash away non-covalently bound protein
(eluate: sample C). My PAGE shows, that there is much less fusion
protein present in sample B than in A. This would suggest a good
coupling efficiency. Unfortunatly, it seems that the Glycine/HCl
treatment results in washing away the coupled protein because sample C
shows high protein content again. How can boiling in laemmli-buffer be
less strong than Glycine/HCl treatment ?
Any comments and suggestions are wellcome.



Ralf R. Weigel

Albrecht von Haller Institute of Plant Sciences
General and Developmental Plant Physiology
Untere Karspuele 2
37073 Goettingen

Tel.:  0551/3919664
Fax: 0551/397820

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