I did protein extraction and purification with ADH_pET11a in BL21. The picture of SDS-Page have total and purified ADH bands shows under 35kda. I want to know why band is under 35 . Expected size is 35.
Dear Yunjin Park
ADH is alcohol dehydrogenase? human form?
do you have an non induced sample?
the presence in the elute of a small band at the correct MW and other more strong band at lower MW, may suggest that you have some protein degradation during the culture.
To confirm this you can:
1) PErform a WB of the elute with policlonal antinodies against your protein (if those are commercially avaiable) to see if both band are positive
2) Send the gel to digestion and peptide analisys by mass spectrometry
in case of degradation you can try to repeat the expression at lower temperature, decrease the induction time or change the E.coli strain.
best regards
Manuele
Hi,
The ~30 kDa band is in high concentration in all lanes. This leads me to suspect that the band is not part of your protein construct and instead is simply carry-over of native protein from E. coli. If you run an uninduced or untransformed sample of BL21. (I did a quick search of other SDS-PAGE gels and found that this protein is common in lysates with other constructs, see the red arrow on the picture attached)
How to get rid of the band?
-Try a more stringent wash protocol
-Switch the affinity resin from Ni-NTA to Co-NTA for more specificity
-Use less affinity resin to try to saturate the binding sites with your protein
-Change your vector to one that uses a His12 tag for tighter binding
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