I am trying to purify a GST tagged protein expressed in HEK T cells. Western Blot has shown that the protein is present in the supernatant, but after purification with glutathione agarose beads (Pierce glutathione superflow kit), the protein can no longer be detected. There is, however, a clear band on the Western Blot for the 'supernatant' from the beads after incubation.
I do not know why my protein does not bind to the beads and was wondering if it could be that cellular glutathione binds the GST tag and prevents binding of the beads.
Any other suggestions are of course also welcome.
Thank you in advance.
Dear Josse Libbrecht ,
There could be several reasons why your GST-tagged protein is not binding to the glutathione agarose beads. Some possible explanations include:
A possible solution to these problems is to optimize the purification conditions and try different buffers or methods. You could also try using a different type of bead, such as a beads that bind to the GST-tag specifically. You can also try to purify in different conditions like increasing or decreasing the pH or salt concentration of the buffer and see if it makes a difference. Additionally, you can try to purify the protein in multiple rounds to increase the yield.
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