Hi everyone
I did GST-pull down exp to test interaction between the GST fusion protein and in vitro translated protein and they interact very strongly with each other. Thus I made double mutation in my protein in the motif that I think is critical for the interaction then repeated the pull down and this mutation disrupted the interaction at all, nice. The problem is I used with wild type buffer with pH 7.5 but when i did the same experiment with the mutant protein I did the whole extraction, binding and washing at pH 8.0. why I did that because the PI for my fusion is 7. Does this make big difference on protein protein interaction in my case?
I run a coomasie gel for GST fusion protein and it is Ok and clean.
Thanks in advance
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laurequillo
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Posted 15 July 2010 - 10:30 PM
saad, on Jul 16 2010, 07:26 AM, said:
Hi everyone
I did GST-pull down exp to test interaction between the GST fusion protein and in vitro translated protein and they interact very strongly with each other. Thus I made double mutation in my protein in the motif that I think is critical for the interaction then repeated the pull down and this mutation disrupted the interaction at all, nice. The problem is I used with wild type buffer with pH 7.5 but when i did the same experiment with the mutant protein I did the whole extraction, binding and washing at pH 8.0. why I did that because the PI for my fusion is 7. Does this make big difference on protein protein interaction in my case?
I run a coomasie gel for GST fusion protein and it is Ok and clean.
Thanks in advance
I did GST-pull down exp to test interaction between the GST fusion protein and in vitro translated protein and they interact very strongly with each other. Thus I made double mutation in my protein in the motif that I think is critical for the interaction then repeated the pull down and this mutation disrupted the interaction at all, nice. The problem is I used with wild type buffer with pH 7.5 but when i did the same experiment with the mutant protein I did the whole extraction, binding and washing at pH 8.0. why I did that because the PI for my fusion is 7. Does this make big difference on protein protein interaction in my case?
I run a coomasie gel for GST fusion protein and it is Ok and clean.
Thanks in advance
Ok, I am not an expert in in vitro interactions, but even when it is true that pH can affect the interaction, I would say that from 7.5 to 8 is not such a big difference, and the disruption is due to the mutation. But if I were you I would repeat it with the same conditions, just to be sure.
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#3
saad
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Posted 15 July 2010 - 10:55 PM
Thanks for your response and good comment.
But I'm wondering if the PI of my fusion 7.0. is it possible to do all stuff( lysis, wash binding..) in buffer with ph7.5? or if I do the lysis and wash in buffer with pH 8.0 then change in pull down exp to buffer with buffer that has pH7.5 is it ok.
Cheers
But I'm wondering if the PI of my fusion 7.0. is it possible to do all stuff( lysis, wash binding..) in buffer with ph7.5? or if I do the lysis and wash in buffer with pH 8.0 then change in pull down exp to buffer with buffer that has pH7.5 is it ok.
Cheers
Edited by saad, 15 July 2010 - 11:00 PM.
