I tried to purified a GST-tagged protein and I used 0.5% triton-x-100 to wash the enriched beads which resulted in a removal of non specific binding to a great extent. I eluted the protein with 50mM glutathione (pH-8) and prepared the samples from elutions and boiled at 100C for 5 minutes. the same I did for the enriched beads after elution to check if the protein is completely eluted or still attached. The eluted samples' CBB showed so many bands while the bead sample has just one band (the expected). I suspected the problem in either boiling or sample buffer, and I change those, but the problem still exists. I have attached the CBB image .
Hameed Akbar Is the beads not washed enough? How did you determine washing is enough? There is insufficient information to say more. For example, what is the expected band, what is the expected molecular weight, and whether the expected band has the appropriate molecular weight, etc. By the way, why is the band of the bead sample "expected"?
@Michitoshi watanabe.. The beads are washed enough as the second lane is more cleaner than the first
which wasnt washed enough. The size of the protein with tag is 86kd and the beads sample shows the correct band size With the correct molecular weight.
how do you know you removed all of the non specific binders? how do you know you don't have native glutathionilated proteins? where are your negative controls?
As David Morgenstern mentioned, what is your negative control?
Also can you run the samples again with a protein ladder for reference?
Is it possible that multiple truncated products of your gene of interest are being generated, which have the GST-tag and as a result they are being eluted as well?
Negative control - IP from the same type of cells, treated the same way, but without your construct
I'm not sure I understand your 2nd question.
re - truncation - theoretically, yes. but that doesn't happen often.
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