I am quantifying the RBM20 in different parts of skeletal muscle from rats. I have six samples , three from tibialis anterior and another three from longissimus dorsi. I tried both GAPDH and Histone for loading control and I loaded 10ul of sample for each well.
When I expose the film I can clearly detect GAPDH, Histone for every samples. But for RBM 20 I just see some fuzzy bands or no bands at all for three samples. When I stain the PVDF membrane with coomassie, I can clearly see strong RBM20 bands for all the six samples. So could anyone help me to analyse this situation? How could I get clear bands on X-ray film?
Hello Maimaiti,
Based on your description, it seems that you have good protein transfer on to the PVDF membranes. Absence of the "specific" band(s) of RBM20 on the x-ray film would suggest that: 1) the primary anti-RBM20 antibody are not suitable for WB analysis (e.g. they may be good for immunocytochemistry, but not suitable for WB), 2) the primary anti-RBM20 antibody are old (i.e. degraded) , 3) secondary antibody used to detect anti-RBM20 antibody are old or poorly interacts with the primary ones.
Solution: 1) try different anti-RBM20 antibody, 2) increase concentration of primary Ab, 3) make sure secondary antibody works well, 4) consider using different blocking/antibody incubation reagent (e.g. BSA vs NP or vice versa).
These suggestion are based on an assumption that your ECL reagents are fresh and work well.
Best of luck
Fuzzy bands on western blots do not necessarily mean immunodetection failour. Fuzzy difuse bands may indicate glycolysation and/or other postranslational modifiication of a target protein. It is important to include a negative control, i.e. a protein sample lacking the RBM20..
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