Western - X-reaction with molecular weight marker (all of a - (Sep/02/2004 )
Im a "newbie" here so hope this hasnt been covered elsewhere.
Basically, the story is that i have made a polyclonal anitbody that has been working pretty well with human muscle extracts, and picks up a couple of bands around the area that i would expect. However, recently one of these bands (approx 70kDa) has been appearing in the molecular weight marker lane after probing. I am using Biorads prestained precision marker. It is a very strong band indeed. It appears to be specific to my antibody, which is causing me great anxiety (it gets personal!!).
I must stress that this has only recently started to occur, i have been working with the Ab for a long time and have never had this problem. The problem cannot be contamination of the ladder as i have run two seperate ladders from different labs!! In conjunction with this, we have noticed that ladders are seperating immmediately in the stack. Now we know all the usual reasons for this (pH of Tris buffers being the major one) and have already thought of most of these, but the problem still occurs???
Does anyone think the two problems are linked? We are having real problems with gels at the moment, things just dont "feel" right.
Any advice would be much appreciated indeed.
The problem is your prestained marker. I am 99% positive that band which you see on your film it is pink band from prestained marker. I don't know reasone, but pink bands from different pre-stained markers often give signal in ECL detection even if you applay no antibody at all