09-02-2010, 05:40 PM
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Join Date: Sep 2010
Location: Lafayette, IN
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| | recombinant histagged protein and western blot
I am expressing histag-proteins and after expression I can easily see a extra band within the correct size of my recombinant protein by SDS-PAGE. However, some of them are negative by western blot (anti-histag). Some are positive (in the same reaction, so the system is working). What can be happening?
Is it possible the any folding problems or inclusion bodies might be interfering with the western results?
I am using:
- For expression:
Gateway (invitrogen) with pDEST 17 (C-terminal 6his) and BL-21 cells.
primary Antibody - mouse anti-His (Invitrogen)
secondary - ant- mouse-HRP
TMB to develop