I have a problem with unspecific bands on my blot.
I have HEK cells transfected with a GFP construct (lane1), mCherry construct (lane2) and RFP construct (lane3). I collect the cells in PBS, do one washing step in PBS, then lyse in RIPA and collect input in SDS buffer. The blot was incubated with an antibody recognizing mCherry and RFP, the constructs are also detected (boxes).
However, in the first lane one can see best the unspecific bands at around 120 kDa and 60 kDa I always get. I read that it could be albumin but since I do already a washing step I thought it should be okay. Next idea would be to do a secondary AB control.
Does anyone have an idea where it can come from? I'm more beginner in terms of western blot.