western blot for membrane protein with his-tag - can't detect with anti-his tag Ab? (Mar/31/2004 )
I am working with purification of a membrane protein with his-tag. As I used 1ml bacterial cell culture and anti-his-tag Ab to detect the expression, I could not see anything. But the positive control which is the protein coming from Ni-NTA column did work. And I know with the expression level, the amount I load should be enough for me to see. Anybody knows any trick about dealing with membrane protein? After I boiled the sample and spin it down, there was a pellet at the bottom of the tube, is it possible that all the protein were still in the pellet after boiling for 10mins? Thanks a lot!
You are detecting protein from Ni-NTA which means the western is working just fine. Your His-Tag protein will have a tendency to stick to chaperones like GroEL and GroES masking the His-tag. 0.1%SDS prevents this interaction. I suggest increasing SDS content of your sample buffer, boiling longer and good luck.
I tried increasing the SDS to 3%(final conc), but the sample ran as a smear on the gel, seems like non-soluble stuff. But I did boil and spin down to get the supernatant. And again no protein is detected by the Ab. Really puzzled.
Consider using a small hydrophobic interaction column like TSK-phenyl to separate some of the crude extract by brute force. We are positive your problem is that of aggregation in crude which is alleviated after His-Tag runs because imidazole would take care of any such associations. We can understand your vexation - you are unable to prove that your starting material has the protein but you end up with it after NiNTA. To prove aggregation in crude, we suggest running a western with just crude extract and increasing amounts of externally added pure protein to these extracts. In parallel, run lanes that have just the pure protein in the same concentration but nothing else. If the crude is masking your protein's His antigenicity, this will be proof because you will get signal with pure protein but a very diminished signal when it is added back into the crude. Also, this experiment might run best with Amersham ECL instead of color reactions
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