Dear all,
I used pET32a to express a transcription factor (TF). The expression and purification went well and i already proved that it's binding to DNA.
Now i'm planning to use the same purified protein to study interation of TF and other protein.
My concern is. Is the binding site to close from N'terminal (only 23 aminoacids apart from Methionine) that Trx will block the interaction TF-protein? (see scheme in attach)
Can anyone help me with this? Do you have any alternative? I can not remove Trx tag because i also have His-tag at the end of my recombinant protein.
If the linker between Trx and the TF has a specific protease cleavage site (TEV, thrombin?), you can do the cleavage and remove the Trx by passing it over the IMAC column, recovering the TF in the flow-through.
Hi Adam,
in fact there is a protease cleavage site, but the problem is that i also have His-tag at the end of my recombinant protein... Thanks for you suggestion anyway
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