western blot protocols, troubleshooting, scientific resources and research atricles for the research community and science students

The electrophoretic transfer of sample proteins to membranes for immunoblotting occurs after protein separation by SDS-PAGE. Care must be used when working with PVDF (polyvinylidene fluoride) membranes - use forceps and handle membranes by their edges so as not to damage the surface.

1. PubMed: comprehensive database - 15 million citations

2. NIH:the National Institutes of Health

3. UniProt: The world's most comprehensive catalogue of information on proteins

4. Western Blot Procedure Western blot protocol overview.

1. Cut filter paper (typical chromatography paper) in approximately 7 x 20 cm pieces.

2. Cut PVDF membrane to 7 x 20 cm.

3. Pre wet the PVDF membrane using 100% methanol for 10 seconds and immerse in dH2O.

4. Soak the filter pads in PVDF Transfer Buffer.

5. Assemble the membrane sandwich according to the kit instructions.

6. Place the sandwich into the transfer assembly.

7. Fill the transfer tank with PVDF transfer buffer.

8. Remove the cooling block from storage at -20 degrees Celsius; place into transfer apparatus.

9. Place the tank in a refrigerator at 4 degrees Celsius.

10. Run the transfer protocol at 0.1 A (constant amperage) overnight.

*note: higher current and voltage setings may be used, however use caution as the transfer buffer may heat up substantially damaging proteins or equipment