in vitro binding between MBP- and GST-fusions

Yoshiki Habu habu at
Wed Sep 26 08:51:05 EST 2001

I am doing an in vitro binding assay between MBP- and GST-fusion proteins.
After incubation, I pulled down the complex with amylose-resin and analyzed
the presence of the GST-fusion in the pellet by Western with an antibody
against the protein fused to GST. Always I got a band that is apparently
twice of the size of the original GST-fusion. Addition of fresh DTT and/or
heating the sample at low temp (70C) did not change the result. This shift
of the size did not happen when the GST-fusion alone was boiled on
glutathione-beads. There is no additional cross-reacting band in the input
MBP- nor GST-fusions. Does anyone have such experience? Thanks for your help.


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