I had cloned 5 gene of interest in pet46EK/LIC and expressed in E. coli BL21(DE3) (Novagen)
The vector will produce 6x his-tag in the front part of the protein.
I had expressed the protein, run SDS-PAGE, and perform western blot.
I use the KPL HisDetector Western Blot Kit, AP Colorimetric, Cat No 25-00-01 https://www.kpl.com/...heet/250001.pdf
for my his-tagged detection.
However, i do encounter few issues:
1. Consistent background protein detected on ~25kDa
------ Do you experience such problem with BL21(DE3)?
------ The background appeared higher than my protein of interest.
2. Inconsistent background protein detected at ~32kDa, only in UREA treated lysate, B.
3. Possible overlapped Recombinant 2 with background on lane B, Recombinant 2?
The PDF I attached had complete descriptions about my problem.
Perhaps you might have an idea on where goes wrong. I really need help in troubleshooting as I got no idea what went wrong...
Million thanks in advance.
Edited by adrian kohsf, 21 March 2011 - 08:11 PM.