I wanted to check translocon proteins (Sec61 alpha, beta, gamma) in endoplasmic reticulm. I got band for alpha subunit protein. However I could not band for beta sub-unit (15 kDa) and gamma sub-unit (7 kDa).
I used following conditions, Gel Running: 16% SDS Gel, 200V, 400mA, 50 Minutes
Blotting Condition: Buffer with 20% ethanol, 80 V/gel, 400 mA, 40 minutes., Nitrocellulose Membrane.
Blocking, 5% Skim Milk, 1 hour.
For such small proteins I suggest using 10/12% tricine-SDS-PAGE, because normal SDS-PAGE (even 15% or more) cannot resolve proteins of this size any more. I'm also working with proteins of this size and for me it works quite nice. You can find the protocol here: doi:10.1038/nprot.2006.4
For the blotting conditions I'm using 20% methanol in the buffer and 40mA per gel for 1h. Nitrocellulose should be fine.
I have been looking at the translocon and use 4-20% gradient gels. I use a normal blotting protocol but fix the blot in 5% acetic acid before blocking. This works well for the beta subunit.
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