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 Molecular Biology
Unit Number:  Unit 10.7
DOI:  10.1002/0471142727.mb1007s84
Online Posting Date:  October, 2008
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Abstract

The staining of proteins bound to a transfer membrane can be useful for determining the efficiency of transfer and marking the location of molecular weight standards. This unit describes three methods for staining blots, using India ink, gold labeling, and fluorescent labeling with SYPRO Ruby. Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for enhancement of staining using alkali treatment. Curr. Protoc. Mol. Biol. 84:10.7.1‐10.7.6. © 2008 by John Wiley & Sons, Inc.

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

     
 
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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
     
 
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Materials

Basic Protocol 1: India Ink Staining

  Materials
  • Protein sample transferred onto blot transfer membrane, nitrocellulose or PVDF (unit 10.8)
  • 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
  • Colloidal gold sol (e.g., Bio‐Rad, Fitzgerald Industries International)
  • Heat‐sealable plastic bag

Support Protocol 1: Alkali Enhancement of Protein Staining

  Materials
  • 1% (w/v) KOH
  • PBS ( appendix 22)
  • Glass or Pyrex dish

Basic Protocol 2: Fluorescent Protein Blot Staining

  Materials
  • Proteins electroblotted onto PVDF or nitrocellulose membrane (unit 10.8)
  • 7% acetic acid/10% methanol
  • SYPRO Ruby protein blot stain (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.
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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 3:2273‐2288.
   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 includes 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.
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