I have attached an image and graphs of the nitrocellulose membrane from the Spot blot assay.
My question is regarding the BM8 lanes.
From the immunofluorescence analysis it shows that cells in serum containing media, treated with puromycin for 30 minutes have a higher translational capacity compared to cells in serum-free media for 30 minutes. This result has been consistent throughout both bone-marrow MSC lines (BM7 and BM8).
The same is confirmed from the spots on BM7 lanes (Lanes 1-4) on the nitrocellulose membrane, however this is not the case for the BM8 spots(Lanes 5-8). What could be the reason for this?
Many thanks
Do you have a "binding control", for example an antibody against a protein that should not be influenced by any treatment? You could use that for normalisation. I guess the binding to the membrane is the problem, not the antigen/antibody. But it's just a guess.
Hi Henna,
I would agree with both Vivica and Sergi above.
sometimes the same membrane may not me suitable for all assays. Also, are you confident your membrane was equilibrated properly before the spotting? this could affect the binding resulting in uneven binding or even high background. In addition you should always include in your blots at least 2-3 positive and 2-3 negative spots for the proteins you are interested in and finally probing with house keeping proteins such as GapDH, tubulin or actin to list a few although i would double check if any of these proteins maybe affected by your experimental conditions.
good luck
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