Advertisement

A Single-Step Simultaneous Protein Staining Procedure for Polyacrylamide Gels and Nitrocellulose Membranes by Alta During Western Blot Analysis

  • Jayanta K. Pal
  • Sunil K. Berwal
  • Rupali N. Soni
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1853)

Abstract

A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS)–polyacrylamide gels and nitrocellulose membranes by Alta during Western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water, viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for Western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining) and cost-effective.

Key words

SDS–polyacrylamide gels Nitrocellulose membrane Western blotting Alta Protein stain 

Notes

Acknowledgment

This work is supported by a research grant from the Department of Atomic Energy, Govt. of India, Bhabha Atomic Research Centre, Mumbai (BRNS Grant number 2007/37166/BRNS) and a grant from the Department of Science and Technology (DST), Govt. of India under the DST PURSE Programme. We wish to thank Dr. Jayant Londhe for his assistance in initiating some preliminary experiments.

References

  1. 1.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  2. 2.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Pal JK, Sarkar A, Katoch B (2001) Detergent-mediated destaining of Coomassie brilliant blue-stained SDS polyacrylamide gels. Indian J Exp Biol 39:95–97PubMedGoogle Scholar
  4. 4.
    Pal JK, Godbole D, Sharma K (2004) Staining of proteins on SDS polyacrylamide gels and on nitrocellulose membranes by alta, a colour used as a cosmetic. J Biochem Biophys Methods 61:339–347CrossRefPubMedGoogle Scholar
  5. 5.
    Kurien BT, Scofield RH (1998) Heat mediated quick Coomassie blue protein staining and destaining of SDS PAGE gels. Indian J Biochem Biophys 35:385–389PubMedGoogle Scholar
  6. 6.
    Merril CR, Goldman D, Sedman SA et al (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211:1437–1438CrossRefPubMedGoogle Scholar
  7. 7.
    Zehr BD, Savin TJ, Hall RE (1989) A one-step low background Coomassie staining procedure for polyacrylamide gels. Anal Biochem 182:157–159CrossRefPubMedGoogle Scholar
  8. 8.
    Wu W, Welsh MJ (1996) Rapid Coomassie blue staining and destaining of polyacrylamide gels. BioTechniques 20:386–388CrossRefPubMedGoogle Scholar
  9. 9.
    Wu M, Stockley PG, Martin WJ II (2002) An improved Western blotting technique effectively reduces background. Electrophoresis 23:2373–2376CrossRefPubMedGoogle Scholar
  10. 10.
    Antharavally BS, Carter B, Bell PA et al (2004) A high-affinity reversible protein stain for western blots. Anal Biochem 329:276–280CrossRefPubMedGoogle Scholar
  11. 11.
    Steinberg TH (2009) Protein gel staining methods: an introduction and overview. Methods Enzymol 463:541–563CrossRefPubMedGoogle Scholar
  12. 12.
    Bajaj AK, Pandey RK, Misra K et al (1998) Contact depigmentation caused by an azo dye in alta. Contact Dermatisis 38:189–193CrossRefGoogle Scholar
  13. 13.
    Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Jayanta K. Pal
    • 1
    • 2
  • Sunil K. Berwal
    • 1
  • Rupali N. Soni
    • 1
  1. 1.Cell and Molecular Biology Laboratory, Department of BiotechnologyUniversity of PunePuneIndia
  2. 2.Dr. D.Y. Patil Biotechnology & Bioinformatics InstituteDr. D.Y. Patil VidyapeethPuneIndia

Personalised recommendations