Pre-Immunodetection Membrane Staining

Pitfalls to avoid:
- Make sure that the stain is compatible with your choice of membrane
- Be certain that the stain does not interfere with antigen:antibody binding
- If the stain is reversible, be sure that the chemistry does not affect antigen epitopes in a detrimental way

For pre-labeling strategies, consider that:
- Both methods couple fluorescent dyes to sample proteins for visualization
- These methods are sensitive to biological variability because labeling is directed against a specific amino acid

Chemical modification of epitopes can introduce variability because:
- Labeling can be variable due to amino acid composition of sample proteins
- It can interfere with electrophoresis or transfer
- It may inhibit antibody binding
Overall, normalization by total protein staining is a very relevant loading control that reduces the impact of biological variability, especially when applied as staining of membranes after transfer.

References
Bio-Rad Bulletin 5781 rev A (2008).

Eaton SL, Roche SL, Llavero Hurtado M, Oldknow KJ, Farquharson C, Gillingwater TH, and Wishart TM (2013) Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.PLoS ONE 8(8): e72457.

Luo S, Wehr NB, and Levine RL (2006) Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green.Anal Biochem. ;350(2): 233-38.

Moritz CP, Marz SX, Reiss R, Schulenborg T, and Friauf E (2014) Epicocconone staining: a powerful loading control for Western blots. Proteomics 14: 162-68.

Collins MA, An J, Peller D, and Bowser R (2015) Total protein is an effective loading control for cerebrospinal fluid western blots. J Neurosci Methods 251: 72-82.

Miller I, Crawford J and Gianazza E (2006) Protein stains for proteomics applications: which, when, why? Proteomics 6(20): 5385-408.

Welinder C and Ekblad L (2011) Coomassie staining as loading control in Western blot analysis. J Prot Res. 10(3): 1416-19.

Romero-Calvo I, Ocón B, Martinez-Moya P, Suárez MD, Zarzuelo A, Martinez-Augustin O, de Median FS (2010) Reversible Ponceau staining as a loading control alternative to actin in Western blots. Anal Biochem 401(2): 318-20.

Gilda JE and Gomes AV (2013) Stain-Free total protein staining is a superior loading control to beta-actin for Western blots. Anal Biochem. 440: 186-88.

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 10(8): e0135392.

Taylor SC, Berkelman T, Yadav G, and Hammond M (2013) A defined methodology for reliable quantification of Western blot data. Mol Biotechnol55: 217-226.

Janes KA (2015) An analysis of critical factors for quantitative immunoblotting.Sci Signaling8(371): rs2.

Gomes R, Gilda JE and Gomes AV (2014) The necessity of and strategies for improving confidence in the accuracy of Western blots.Expert Reviews Proteomics11(5): 549-60.

Taylor SC and Posch A (2014) The design of a quantitative Western blot experiment.BioMed Res Intl. doi: 10.1155/2014/361590.

Degasperi A, Birtwistle MR, Volinsky N, Rauch J, Kolch W, Kholodenko BN (2014) Evaluating strategies to normalise biological replicates of Western blot data.PLoS ONE 9(1): e87293.

About LICORbio
LICORbio offers imaging systems, analysis software, and IRDye® infrared dye reagents for quantitative protein and small animal imaging. LICORbio provides an integrated solution for quantitative Western blots with its Odyssey® Infrared Imaging Systems, Image Studio™ quantification software, Empiria Studio® analysis software for publication-ready data, reagents, unique IRDye® secondary antibodies, and online training courses.

For chemiluminescent Western blots, the C-DiGit® Blot Scanner is a personal, portable imager. Use the Odyssey® XF or Odyssey M Imaging Systems for both chemiluminescence and near-infrared fluorescence Western blots. You can achieve superior small animal imaging results using BrightSite™ near-infrared fluorescent probes or bioluminescence on the Pearl® Trilogy imaging system.

LICORbio Website
[ Ссылка ]

Reagent Store
[ Ссылка ]

Image Studio™ Software
[ Ссылка ]

LinkedIn
[ Ссылка ]

Twitter
[ Ссылка ]

Facebook
[ Ссылка ]

свернуть