Hello,
you probably purified a contaminant. Do a blot with an anti-biotin
antibody or get electro-spray mass spectrometry done in order to
confirm the identity of your protein.
Wim
On 13/11/2018 11:13, Anamika Singh
wrote:
I am purifying the biotinylated protein (cloned into the pET28a vector) using Avidin beads. Since I need the protein for SPR but when I used the purified protein to interact with Streptavidin coated onto the SPR chip. There was no signal. Can anybody tell me why is it so or how can I make sure that the purified protein is biotinylated enough to interact and give the signal? Because when I ran the SDS-PAGE there was a band of purified protein. I have used the elution buffer (20mM Tris, 5mM Mgcl2, 500mM Nacl, and 2mM Biotin). PS: I have included the biotin during overexpression of the protein also.
Please suggest.
Thanks --
Anamika
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel
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--
Wim Burmeister
Professeur
Institut de Biologie Structurale (IBS) CIBB
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