Help! Protein extract degradation ? - (May/01/2006 )
I am having bizarre things happening to my lysates, basically I am extracting protein form ~ 2X10^6 Hodgkins cell lines, one of the lysates which when quantified appears to be in abundance i.e after A595 reading of 3 repeats. Once run on a western and I blot for actin, that sample has very small amounts compared to other samples, there are no smears though and so I am confused as to whether this is due to degradation or what ?infact as I am loading 25ug of protein, the actin is all over the place! does the fact that I get absolutely loads of protein affect their quantitation on the spec? I am truelly baffled and would welcome any suggestion/ help in this regards, many thanks
I think there are two possibilities, one is that the well of the gel is not good, this maybe caused by the gel which is not sealed tightly (the two glasses) when you make it. Another one is the transfer process. Did you stir your transfer buffer if you used wet transfer?