Interaction analysis
Ankan Banerjee
Hello,
I am trying to establish interaction of a protein with its predicted neighbours, required for a complex assembly. I have my bait proteinwith His-GST tagged(N terminal) and another protein with only His tag fusion. I could able to purify both of the proteins using Ni-NTAcolumn. Now I tried GST resin,Clonetech for interaction experiment. My idea was to bind the HIS-GST protein to the GST resin and then flow the HIS tagged protein through it. Whetherif they are interacting then these two proteins should elute together. But I got a strange result, First when i tried the experiment then it seems like they are interacting then i proceed for control experiments, I flow only the His tagged protein through GST resin and some how my protein bind to the GST resin (which should not be the case). For your kind information I must mention that my His tagged protein has high tendency to form large oligomer(we still could not able to confirm how big..!).
Can anyone please suggest me why this His tagged protein is giving such a scenario?
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ANKAN BANERJEE
tanmay meher
please specify your question properly and correctly, I am not able to
understand it, and by the way what is all this links, it is directing
to wikipedia....
sun frenk
structure of the protein it self which could bind to the GST beads.
Mean while, binding and wash buffer may be very important for your
experiment, to elute nonspecific binding, you may add low
concentration of reduced GSH in the buffer. Best wishes.
2011/6/27 tanmay meher <meher....@gmail.com>:
>
> please specify your question properly and correctly, I am not able to
> understand it, and by the way what is all this links, it is directing
> to wikipedia....
>
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Haibo Sun
Nanjing Medical University
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