Hi everyone!
I try to detect VDR protein (~48 kDa) using Western Blot. After SDS-PAGE using 10% MP TGX Stain-Free Gel from BioRad I transferred the protein onto an metOH activated PVDF membrane using the TurboTransfer System from BioRad. After blocking the membrane with EveryBlot Blocking Buffer (BioRad) for 5 minutes (according to protocol) I incubate with primary AB (Santa Cruz, 1:500, see figure blot 1) at 4 degress overnight. After wasching 3x with PBS-T (0.05% tween-20), membranes were incubated for 5 hours at room temperature with secondary AB ( m-IgGκ BP-HRP, 1:5000). Both antibodies were diluted in the EveryBlot Blocking Buffer. Chemiluminescence was detected using ECL solution (1 minute incubation). Autoexposure was 17 minutes (possible reason?).
I know reducing background several options exist: reducing AB concentration, incubation time; increasing blocking time, and so on.
Since there are so many possibilities to reduce background, maybe you have suggestions which work most efficiently.
Another membrane (run in parallel) I incubated with primary AB from R&D. There I have another problem: lot of unspecific bands and no specific band at 48 kDA. (see figure blot 2)
Thank you very much in advance.
Hey Christian Behm
Did you stain your membranes with ponceau?
The membrane 1 appears to have some issues in the transfer.
Hey
Thank you for your rapid reply. We checked transfer/blot by StrainFree Imaging Technology from BioRad. It looked good. Transfer does not seem to be the problem.
Could it be acculumated ECL solutation? Or an issue with antibodies, washing or blocking? I thought I will increase blocking time and washing steps.
Thank you very much.
Maybe you should consider using standard gels for your western blot transfer or skip the UV-detection step before the western blot. Note, that the stain-free gels require a UV-induced chemical modification of some amino acid side chains to become visible by fluorescence in the gel. If your antibodies bind to modified epitopes or close to the modified side chain, it could at least lead to a reduced signal intensitiy (because a fraction of the epitope could be modified or binding could be weaker) or loss of signal.
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