Western Blotting
SDS-PAGE: If protein of interest is expressed at very low levels in the cells and/or only a very small percentage of cells in the tissue express the desired protein, then a higher amount of total tissue lysate or crude membrane lysate in the range of 100 to 150 ug of protein per lane is recommended. SDS-PAGE gels used should contain appropriate percent acrylamide to provide the best separation of proteins in the molecular weight range of the desired protein to be detected. We strongly recommend separating proteins on SDS-PAGE gels at 15 to 20 mA constant current when 100 to 150 ug of protein is loaded in each lane to ensure clear separation. SDS-PAGE can be run overnight at 5 to 7 mA constant current and next day in the morning the current should be increased to 20 mA until the dye front reaches the bottom of the gel.

Transfer of Proteins to Nitrocellulose: When transferring hydrophobic integral cell membrane proteins Tris-Glycine buffer containing 0.1 % SDS running buffer with 10 to 20 % methanol should be used to facilitate transfer of proteins like chemokine receptors that have 7 hydrophobic domains. Tris-Glycine buffer with 10 to 20 % methanol is adequate to transfer most secreted proteins. Transfer the proteins following the instructions provided by the manufacturer of the transfer apparatus. For the semi-dry transfer system we used, protein transfer was done at 150 mA constant current for 60 to 90 minutes (min).

Immunodetection: After the transfer, air dry the nitrocellulose membrane on a paper towel. Rinse the membrane in Tris buffered saline with 0.1 % Tween 20 (20 mM Tris, 150 mM NaCl pH 7.5 to 8.0) for 5 min. Block the membrane in 5 % milk in TBS-Tween for a minimum of 1 hour. Incubate with primary antibody diluted in 1 to 2% milk in TBS-Tween at RT for 2 hours or overnight at 4 C. Wash the membrane x 5, 5 min each with TBS-Tween containing 300 mM NaCl (Note: TBS-Tween indicated above contains 150 mM NaCl. Higher salt is used to facilitate removal of non-specifically bound antibody). Incubate with HRP conjugated secondary antibody at appropriate dilution in 1 to 2 % milk in TBS-Tween containing 150 mM NaCl for a minimum of 1 hour. If using B cells the secondary antibody should be species specific to minimize detection of Ig expressed by B cells by secondary antibody. Wash the membrane x 5, 5 min each with TBS-Tween containing 300 mM NaCl. Visualize the bands using the ECL system of Amersham-Pharmacia.

If you have an questions or concerns with regards to these protocols please contact us.

The majority of our research antibodies originated from collaborations using our high quality, reliable, and traceable polyclonal antibody services provided in goats, sheep, and rabbits. Many valuable antibodies have been produced at Capralogics as verified by a long history of peer reviewed scientific publications.

Contact Capralogics
antibodyinfo@capralogics.com
FAX 1-413-643-0067
Phone 1-800-975-6866