Western blotting in my laboratory is not currently working well. I think we need to overhaul it. I was wondering if anyone let us know which transfer system (company and machine name) is most established and recommended in the field. Our machine (not disclosing) is not the best one. In addition, I would like to know what substrate you use for detection of bands.
Thank the community in advance
Ken
Hey Kensuke,
we are using the Bio-Rad SemiDry Blotting System.
We perform SDS-PAGE with the protocol of the Bio-Rad Laboratories, Inc. by using a Criterion Gel cassette containing a 12 % Tris/HCl gel mounted in a Criterion Cell filled with 1X TGS running buffer. Lysates were heated for protein denaturation for 10 min at 95 °C, incubated on ice for SDS-PAGE is performed at a constant voltage of 70 V.
The Gels are then blotted onto a nitrocellulose membrane (0.1 µm, Amserham) using a Trans-Blot® Turbo™ Transfer system (Bio-Rad). Blotting was performed with the StandardSD protocol (constant 25 V, 1 A, 30 min) in accordance to the BioRad instructions.
We incubate membranes after antobody staining and washing in Amserham ELC detection reagents (GE Healthcare) for 1 min and detect protein bands with a ChemiDocTM (Bio-Rad Laboratories, Inc.).
I hope that helps you.
Please feel free to ask any further questions.
All the best and stay healthy,
Marc
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