Western blot analysis of Na+/Ca2+ channels has several crucial stages. Please see the following specific protocol for that channel.
Tissue preparation: From our experience, in order to avoid degradations during membrane preparation a protocol that does not use detergent should be used (resulting with membrane fraction rather than lysate). Using such protocol minimizes the degree of degradation (the protocol is available on our website)
Running the gel: Channels above 150 kDa, we recommend using a 4-6% gel.
Before loading, heat the samples in sample buffer at 70ºC for 10 min (again, to minimize protein degradations).
Transfer: The transfer stage is crucial. The recommended conditions for the transfer are as follows: 100 mA for 22 h at 4ºC.
Western blotting: Block the membrane for 2-5 hours with 3% BSA or 5% skim milk with 0.05% NaN3 with 0.1%Tween-20. Apply the primary antibody (in blocking solution) for overnight incubation at 4ºC with gentle agitation (1:200). After the incubation wash 3 times with TBS-T (0.1% Tween 20) or PBS-T.
From this stage on, do not use any NaN3!
Apply secondary antibody (goat anti rabbit IgG) in blocking solution without NaN3 at the recommended dilution for 1 h at room temperature with gentle agitation.
ECL: We are using a commercial kit.
Exposure to film: Depending on the abundance of the channel, the exposure time may vary from 1 min – 1 h in order to detect the the channel.