Acrylamide percentage varies in SDS-PAGE gels. Use lower percentage acrylamide when immunoblotting high molecular weight proteins and higher percentage acrylamide when immunoblotting low molecular weight proteins.
This protocol uses a dry transfer device but can be adapted for wet and semi-dry transfer approaches.
Day 1: Prepare lysates, run SDS-PAGE, transfer, block, incubate with primary antibody
Day 2: Incubate with secondary antibody
Tips and Troubleshooting:
The optimal lysis buffer will vary depending on the sample type and cellular location of the protein of interest. You may need to try a variety of lysis buffers to find the best for your target.
The optimal concentration and blocking buffer will vary between antibodies. Review the instructions before starting your experiment and consider titrating your antibody to determine the optimal dose.
To ensure that your antibody is both functioning as expected and is specific, include a positive control sample that you know expresses the protein, such as cells transfected with the protein of interest, and a negative control sample that does not express the protein of interest.