A defined methodology for reliable quantification of western blot data

Molecular Biotechnology

Volumn 55, Issue 3, 2013, Pages 217-226

  Taylor, Sean C ^a   Berkelman, Thomas ^b   Yadav, Geetha ^b   Hammond, Matt ^b  

^a Bio Rad Laboratories (Canada) Ltd   (Canada)

^b BIO RAD LABORATORIES   (United States)

Author keywords

Densitometry; Protein expression; Western blot

Indexed keywords

ANALYSIS METHOD; DENSITOMETRY; DETECTION METHODS; PROTEIN EXPRESSIONS; QUANTITATIVE DATA; QUANTITATIVE METHOD; WESTERN BLOTS; WESTERN BLOTTING;

CHEMILUMINESCENCE;

PROTEINS;

POLYVINYLIDENE FLUORIDE; PROTEIN;

ANALYTIC METHOD; ARTICLE; AUTOFLUORESCENCE; CHEMOLUMINESCENCE; DIGITAL IMAGING; LIMIT OF DETECTION; LIMIT OF QUANTITATION; LINEAR DYNAMIC RANGE; MEMBRANE TRANSPORT; METHODOLOGY; OUTCOME ASSESSMENT; POLYACRYLAMIDE GEL ELECTROPHORESIS; PROTEIN ANALYSIS; PROTEIN EXPRESSION; PROTEIN TRANSPORT; QUANTITATIVE ANALYSIS; RELATIVE DENSITY; STANDARDIZATION; ULTRAVIOLET RADIATION; WESTERN BLOTTING;

ANIMALS; BLOTTING, WESTERN; DENSITOMETRY; HELA CELLS; HUMANS; LUMINESCENT MEASUREMENTS; PROTEINS; REFERENCE STANDARDS; REPRODUCIBILITY OF RESULTS;

EID: 84890553910     PISSN: 10736085     EISSN: 15590305     Source Type: Journal    
DOI: 10.1007/s12033-013-9672-6     Document Type: Article

References (22)

1. Towbin, H.; Staehelin, T.; & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. PNAS, 76(9), 4350-4354.
2. Bio-Rad Laboratories (2011) Protein Blotting Guide, Interactive PDF. Review B, Bulletin 2895.
3. Lin, J. C.; Choi, E. I.; & Pagano, J. S. (1985). Qualitative and quantitative analyses of Epstein-Barr virus early antigen diffuse component by western blotting enzyme-linked immunosorbent assay with a monoclonal antibody. Journal of Virology, 53, 793.
4. Kurien, B. T.; & Scofield, R. H. (2006). Western blotting. Methods, 38(4), 283-293.
5. Editorial. (2006). A picture worth a thousand words (of explanation). Nature Methods, 3(4), 237.
6. Schiavini, D. G.; Puel, J. M.; Averous, S. A.; & Bazex, J. A. (1989). Quantitative western immunoblotting analysis in survey of human immunodeficiency virus-seropositive patients. Journal of Clinical Microbiology, 27(9), 2062-2066.
7. Elbaggari, A. McDonald, K.; Alburo, A.; Martin, C. (2008) Imaging of chemiluminescent western blots: comparison of digital imaging and X-ray film. Bio-Rad Tech Note 5809.
8. Suzuki, O.; Koura, M.; Noguchi, Y.; Uchio-Yamada, K.; & Matsuda, J. (2011). Use of sample mixtures for standard curve creation in quantitative western blots. Experimental Animals, 60(2), 193-196.
9. Mollica, J. P.; Oakhill, J. S.; Lamb, G. D.; & Murphy, R. M. (2009). Are genuine changes in protein expression being overlooked? Reassessing western blotting. Analytical Biochemistry, 386, 270-275.
10. Dubitsky, A. and Perreault, S. A Model for Binding of DNA and Proteins to Transfer Membranes. Pall Corporation Tech Note.
11. Plant, A. L.; Locascio-Brown, L.; Haller, W.; & Durst, R. A. (1991). Immobilization of binding proteins on nonporous supports. Applied Biochemistry and Biotechnology, 30, 83-97.
12. Dittmer, A.; & Dittmer, J. (2006). Beta-actin is not a reliable loading control in Western blot analysis. Electrophoresis, 27(14), 2844-2845.
13. Aldridge, G. M.; Podrebarac, D. M.; Greenough, W. T.; & Weiler, I. J. (2008). The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting. Journal of Neuroscience Methods, 172(2), 250-254.
14. Rocha-Martins, M.; Njaine, B.; & Silveira, M. S. (2012). Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and western blot throughout rat retinal development. PLoS One, 7(8), e43028.
15. Lanoix, D.; St-Pierre, J.; Lacasse, A. A.; Viau, M.; Lafond, J.; & Vaillancourt, C. (2012). Stability of reference proteins in human placenta: general protein stains are the benchmark. Placenta, 33(3), 151-156.
16. Greer, S.; Honeywell, R.; Geletu, M.; Arulanandam, R.; & Raptis, L. (2010). Housekeeping genes; expression levels may change with density of cultured cells. Journal of Immunological Methods, 355(1-2), 76-79.
17. Ferguson, R. E.; Carroll, H. P.; Harris, A.; Maher, E. R.; Selby, P. J.; & Banks, R. E. (2005). Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies. Proteomics, 5(2), 566-571.
18. Colella, A. D.; Chegenii, N.; Tea, M. N.; Gibbins, I. L.; Williams, K. A.; & Chataway, T. K. (2012). Comparison of stain-free gels with traditional immunoblot loading control methodology. Analytical Biochemistry, 430(2), 108-110.
19. Gürtler, A.; Kunz, N.; Gomolka, M.; Hornhardt, S.; Friedl, A. A.; McDonald, K.; et al. (2013). Stain-free technology as normalization tool in western blot analysis. Analytical Biochemistry, 433(2), 105-111.
20. Romero-Calvo, I.; Ocón, B.; Martínez-Moya, P.; Suárez, M. D.; Zarzuelo, A.; Martínez-Augustin, O.; et al. (2010). Reversible Ponceau staining as a loading control alternative to actin in Western blots. Analytical Biochemistry, 401(2), 318-320.
21. Welinder, C.; & Ekblad, L. (2011). Coomassie staining as loading control in Western blot analysis. Journal of Proteome Research, 10(3), 1416-1419.
22. Hagiwara, M.; Kobayashi, K.; Tadokoro, T.; & Yamamoto, Y. (2010). Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis. Analytical Biochemistry, 397(2), 262-264.