I had made BL21(DE3) cell competent.And for ensure that i had done transformation which gave many many colonies.So i start to do protein expression with this cell.But I can't understand it is not showing proper band in gel.
At first there was a sonication problem then we reduce power and time.
So can anyone tell me what are the criteria of a cell to let itself for express a protein properly.
I used 100 ml culture,then 10 ml lysis buffer,sonicate for 3 mins for 10 sec on 30 sec off
A key detail here may be how you are growing and harvesting the cells - in other words, the steps upstream or prior to sonication. Are you inducing expression at a mid-log OD with IPTG, and if so how much and for how long?
@Devin Camenares Secondery culture had been growing until O.D 0.65(actual value should be 0.6 though, there was an error it become 0.65).Then it was kept at 18 degree for 30 mins,after that it was induced with 1mM IPTG for 20.5 hours.
Have you tried loading s whole cell lysate, pre-sonication? Just pellet some cells, resuspend in gel loading buffer, boil and load.
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