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recombinant histagged protein and western blot

recombinant histagged protein and western blot - Western Blot Forum

recombinant histagged protein and western blot - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 09-02-2010, 05:40 PM
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Question recombinant histagged protein and western blot



Hello all,

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.
- Western:
primary Antibody - mouse anti-His (Invitrogen)
secondary - ant- mouse-HRP
TMB to develop
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blot , gateway , histag , histagged , protein , recombinant , recombinant proteins , western , western blot


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