Detection of Proteins on Blot Transfer Membranes

Joachim Sasse1, Sean R. Gallagher2

1 Shriners Hospital for Crippled Children, Tampa, Florida, 2 UVP, Inc., Upland, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.10B
DOI:  10.1002/0471142735.im0810bs83
Online Posting Date:  November, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one‐dimensional or two‐dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. Protocols are provided for the use of six general protein stains: Amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, and India ink. In addition, the fluorescent stains fluorescamine and IAEDANS, which covalently react with bound proteins, are described. Approximate detection limits for each nonfluorescent stain are indicated along with membrane compatibilities. Curr. Protoc. Immunol. 83:8.10B.1‐8.10B.6. © 2008 by John Wiley & Sons, Inc.

Keywords: nylon; nitrocellulose; PVDF; protein/blotting; stain; total protein; fluorescence

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: India Ink Staining
  • Alternate Protocol 1: Gold Staining
  • Support Protocol 1: Alkali Enhancement of Protein Staining
  • Basic Protocol 2: Fluorescent Protein Blot Staining
  • Support Protocol 2: Viewing and Photographing the Blot
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: India Ink Staining

  Materials
  • Protein sample transferred onto blot transfer membrane, nitrocellulose or PVDF (unit 8.10)
  • Tween 20 solution (see recipe)
  • India ink solution (see recipe)
  • Plastic boxes
NOTE: Deionized, distilled water should be used throughout this protocol.

Alternate Protocol 1: Gold Staining

  Materials
  • Membragold colloidal gold solution (Bio‐Rad, Fitzgerald Industries International)
  • Heat‐sealable plastic bag

Support Protocol 1: Alkali Enhancement of Protein Staining

  Materials
  • 1% (w/v) KOH
  • PBS ( appendix 2A)
  • Glass or Pyrex dish

Basic Protocol 2: Fluorescent Protein Blot Staining

  Materials
  • Proteins electroblotted onto PVDF or nitrocellulose membrane (unit 8.10)
  • 7% acetic acid/10% methanol
  • SYPRO Ruby protein blot stain (purchase from Invitrogen/Molecular Probes; also see recipe)
  • Small polypropylene staining dish
  • Orbital shaker
NOTE: Perform all washing, staining, and other incubation steps with continuous, gentle agitation (e.g., on an orbital shaker at 50 rpm). For PVDF membranes, be sure to float the membrane face down on the solution.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Antharavally, B.S., Carter, B., Bell, P.A., and Krishna Mallia, A. 2004. A high‐affinity reversible protein stain for western blots. Anal. Biochem. 329:276‐280.
   Hancock, K. and Tsang, V.C.M. 1983. India ink staining of proteins on nitrocellulose paper. Anal. Biochem. 133:157‐162.
  Mackintosh, J.A., Choi, H.Y., Bae, S.H., Veal, D.A., Bell, P.J., Ferrari, B.C., Van Dyk, D.D., Verrills, N.M., Paik, Y.K., and Karuso, P. 2003. A fluorescent natural product for ultra sensitive detection of proteins in 1‐D and 2‐D gel electrophoresis. Proteomics 145:315‐321.
   Moeremans, M., Daneels, G., and De Mey, J. 1985. Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes. Anal. Biochem. 145:315‐321.
   Sutherland, M.W. and Skerritt, J.H. 1986. Alkali enhancement of protein staining on nitrocellulose. Electrophoresis 7:401‐406.
Key References
   Moeremans et al., 1985. See above.
  For investigators wishing to prepare their own reagents, this paper include descriptions of how to make gold and iron sols.
   Patton, W.F. 2000. A thousand points of light: The application of fluorescence detection technologies to two‐dimensional gel electrophoresis and proteomics. Electrophoresis 21:1123‐1144.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library