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.
Hi Marit
I have also been encountering similar artefacts in my WB, albeit with different primary antibody. It could be a non-specific band from the secondary antibody.
If it was albumin, you would see it in all lanes right?
Could you please specify the size of the protein in the mCherry lane? with and without the tag? What antibody are you using to detect the protein of interest here?
Hope you manage to solve this soon
Best
Aparajita
The "m" in mCherry stands for monomeric because the protein has been engineered to not form dimers like natural fluorescent proteins tend to do.
I think the lower band in lane 2 is the Cherry monomer, while the higher band in lane 2 and the other lanes are dimers and higher-order multimers. They're still functional proteins, they just behave differently in situ
Hi Marit, if you are only using a primary antibody, then based on your statement that ln 1 is gfp, lane 2 is McHenry and lane 3 is rfp transfected, i will assume that the other bands are serum proteins. You should repeat the wb with a non transfected control to confirm. If that is also positive then you should purchase a poly antibody t
against McHenry that has been affinity purified and assessed for xr against serum proteins. I would also recommend confirming your initial wb results by flow cutomtery.
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