1.
Sample Prepartion is critical.
Garbage samples gives garbage results.
2.
The quality of your polyacryamide gels and running buffers can affect the quality of your protein separation
3.
Many improvements in the transfer of proteins from gel to membrane have occured in the last 5 years. Use an apparatus such as the Turbo Blot for faster (7 min) transfer.
4.
Use total protein normalization, which is often better than a using a single housekeeping protein. We use Stains Free or Ponceau S staining.
5.
Optimize the blocking and primary antibody amounts for antibodies that will be used regularly. Don't forget to validate that the antibody specifically binds to the protein of interest.
6.
Take multiple blot exposures irrespective of whether using chemiluminesce or fluorescence. Newer machines show when bands are overexposed.
7.
Enjoy your beautiful Western Blotting result.
THE BASICS
A good protocol gives you Western Blots that are picture perfect.
Colorimetric, chemiluminescence, and fluorescence detection are very different and the procedures for each needs to be independently optimized.
WHAT WE USE
These are the equipment we currently use
COMMON MISTAKES
1. Poor Quality or incorrect dilution of Primary
Antibody
2. Poor Quality Sample
3. Bad/Expired Polyacrylamide Gel
4. Poor Quality or incorrect dilution of Secondary Antibody
5. Incorrect Running Buffer
6. Incorrect Transfer Buffer
7. Incorrect or contaminated Tris Buffered Saline (TBS)
8. Overexposure of Chemiluminescent or fluorescent signals
VALIDATING ANTIBODIES
It is critical to use high quality antibodies for reproducible Western Blots.
Some of the Western Blotting Publications in which Dr. Gomes was asked for his expert opinion or as a validation reference
Jan L.A. Voskuil , Anita Bandrowski, A., Begley, C.G., Bradbury, ARM., Chalmers, AD., Gomes, AV., Hardcastle, T., Lund-Johansen, F., Plückthun, A., Roncador, G., Solache, A., Taussig, MJ., Trimmer, J.S., Williams, C., Goodman, S.L. (2020) The Antibody Society’s antibody validation webinar series. mAbs. 12:1, 1794421.
Belisle, C. (2010) Reproducible Laemmli PAGE Performance. Tutorial. Nov. 1, 30:No 19. (Includes Top Ten Tips to Improve Protein Gel Electrophoresis)
Useful Western Blotting References from the Gomes Lab
Mishra M, Tiwari S, Gunaseelan A, Li D, Hammock BD, Gomes AV. (2020) Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP). Electrophoresis, 40(12-13): 1731-1739.
Sander H, Wallace S, Plouse R, Tiwari S, Gomes AV. (2019) Ponceau S waste: Ponceau S staining for total protein normalization. Analytical Biochemistry, 575: 44-53.
Mishra M, Tiwari S, Gomes AV. (2017) Protein purification and analysis: next generation Western blotting techniques. Expert review of proteomics. 14(11):1037-1053.
Gilda JE, Ghosh R, Cheah JX, West TM, Bodine SC, Gomes AV. (2015) Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS). PLoS One. 19;10(8):e0135392.
Gilda JE, Gomes AV. (2015) Western blotting using in-gel protein labeling as a normalization control: stain-free technology. Methods Mol Biol. 1295:381-91
Ghosh R, Gilda JE, Gomes AV. (2014) The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev Proteomics. 11(5):549-60.
Gilda JE, Gomes AV. (2013) Stain-Free total protein staining is a superior loading control to β-actin for Western blots. Anal Biochem.; 440:186-8.