Hi,
I am trying to purify my POI its copurying with a chaperon. so i have decided to do a MgATP wash to get rid of chaperon. But my query is that i am using pottasium phosphate buffer at Ph 7.4 for talon resin . and i have studied in literature that pottasium sequester divalent cations. Now i am planning to do a MgATP wash. will the buffer will decrease the concentration of mg in the reaction. further the other option which people have used with Ni NTA colums is tris buffer at PH8. but tris buffer is not recommended for talon resin though it could be used till 50mM since tris contain primary amines which can interfer with binding. the Pi of my His tag protein is 7.1
Which other parameters could be looked in for protein expression optimization. I already have optimized expression temp with 1mM IPTG. Protein appears on SDS page gel as two band one of protein and other of chaperon
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