Small Scale Bacterial Expression - protein purification (Aug/27/2007 )
I'm new in protein purification. I need to screen the rigth conditions for protein expression in E.coli (induction time). Can I load directly the bacterial pellet boiled in SDS-PAGE loading buffer? or should I sonicate?.... And How much of bacterial culture is enough to screen bacterial expression by western blot? Thanks!!
we always do sonication, after which you can separate the soluble phase from the insoluble phase. Depending on the solubility of your recombinant protein, you will need a different approach to do the purification. If you boil the bacteria, you will only be able to see whether the protein is expressed, not whether it is present in a soluble form or under the form of an inclusion body.
To test a construct for the expression of the recombinant protein, I normally spin down 1,5 ml of the induced culture, and resuspend in 300 µl, but in the end I load 20-25 µl on my gel.
i saw ur reply... iim also working in protein expression and purification.... how can i detect whether my protein is soluble or insoluble.. finally i resuspend in 100ul of SDS-PAGE sample buffer and load 25ul in my gel.. i can noticed bands in desired mol wt.. but i cant get it when i handle purification.... im using Qiagen purification system.. in that gravity flow and denaturing conditions are followed..suggest me..
To determine whether your protein is soluble, lyse the cells by sonication, freeze/thaw or your favourite method, then centrifuge at max speed for 10 minutes to pellet the debris. Collect the supernatant - that is your soluble fraction. Re-suspend the pellet in buffer and collect - this is the insoluble fraction.
yep, that's also the way I do it, although I do add an extra washing step (with MilliQ) of the pellet to remove all of the soluble phase from the insoluble phase