Electroblotting from Polyacrylamide Gels

Aaron Goldman1, Jeanine A. Ursitti1, Jacek Mozdzanowski1, David W. Speicher1

1 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 10.7
DOI:  10.1002/0471140864.ps1007s82
Online Posting Date:  November, 2015
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Abstract

Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N‐terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents for specialized applications. © 2015 by John Wiley & Sons, Inc.

Keywords: electroblotting; electrotransfer; protein transfer; SDS‐PAGE gel transfer; polyacrylamide gel transfer; western blotting

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Electroblotting onto PVDF Membranes
  • Alternate Protocol 1: Electroblotting of Proteins for Sequence Analysis
  • Alternate Protocol 2: Electroblotting onto Nitrocellulose Membranes
  • Alternate Protocol 3: Protein Electroblotting in Semidry Systems
  • Alternate Protocol 4: Protein Electroblotting in Dry Systems
  • Basic Protocol 2: Protein Elution from PVDF Membranes Using Detergents
  • Alternate Protocol 5: Protein Elution from PVDF Membranes Using Acidic Extraction with Organic Solvents
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Electroblotting onto PVDF Membranes

  Materials
  • 1× transfer buffer (see recipe)
  • Polyacrylamide gel containing proteins of interest (units 10.1 10.4)
  • 100% methanol
  • Powder‐free gloves
  • Electroblotting apparatus: “solid” plate electrode tank transfer system (e.g., Trans‐Blot Cell, Bio‐Rad)
  • Glass dishes and trays
  • Gel support sheet (e.g., porous polyethylene sheet, Curtin Matheson)
  • PVDF transfer membrane: Immun‐blot (Bio‐Rad), Immobilon‐P (Millipore)
  • Whatman no. 1 filter paper
  • Power supply (500 V, 300 mA)
  • Additional reagents and equipment for staining gels (unit 10.5)
NOTE: Use powder‐free gloves when handling all materials for this procedure and handle membranes with forceps at the edges to avoid potential artifactual staining.

Alternate Protocol 1: Electroblotting of Proteins for Sequence Analysis

  Additional Materials (also see protocol 1)
  • Thioglycolate (thioglycolic acid, sodium salt; Sigma)
  • 2× or 6× SDS sample buffer (for discontinuous systems; unit 10.1)
  • PVDF transfer membrane: Sequi‐Blot (Bio‐Rad) or Immobilon‐PSQ (Millipore)
  • Additional reagents and equipment for one‐ or two‐dimensional gel electrophoresis (units 10.1 10.4) and for staining membranes (unit 10.8) and gels (unit 10.6)

Alternate Protocol 2: Electroblotting onto Nitrocellulose Membranes

  Additional Materials (also see protocol 1)
  • 0.45‐μm nitrocellulose transfer membrane (Schleicher & Schuell, Bio‐Rad)
  • Additional reagents and equipment for membrane (unit 10.8) and gel staining (unit 10.6)

Alternate Protocol 3: Protein Electroblotting in Semidry Systems

  Additional Materials (also see protocol 1)
  • Semidry transfer apparatus (e.g., Trans‐Blot SD Semidry Transfer Cell, Bio‐Rad)
  • Razor blades
  • Mylar mask (optional)

Alternate Protocol 4: Protein Electroblotting in Dry Systems

  Additional Materials (also see protocol 1)
  • High‐quality water (e.g., Milli‐Q purified water)
  • Razor blades
  • Gel roller
  • Dry transfer apparatus (e.g., iBlot 2, Life Technologies)
  • Proprietary transfer stack kit (e.g., preassembled gel stacks, absorbent pads, and filter paper from Life Technologies)

Basic Protocol 2: Protein Elution from PVDF Membranes Using Detergents

  Materials
  • Polyacrylamide gel containing proteins of interest
  • Ponceau S stain (unit 10.8; optional)
  • 50% methanol (optional)
  • Triton/SDS elution buffer (see recipe)
  • PVDF transfer membrane (Immobilon‐P, Millipore)
  • Transfer apparatus: solid plate electrode tank transfer system (e.g., Trans‐Blot, Bio‐Rad)
  • Plastic wrap
  • Soft pencil
  • Clean razor blade or scalpel
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • Additional reagents and equipment for staining membranes (unit 10.8)

Alternate Protocol 5: Protein Elution from PVDF Membranes Using Acidic Extraction with Organic Solvents

  Additional Materials (also see protocol 6)
  • Extraction solution (see recipe)
  • 50% methanol
  • 1:2 (v/v) acetonitrile/formic acid
  • Aluminum foil dust cover
  • Lyophilizer
NOTE: All solutions should be made with high‐purity water and can be stored at room temperature for at least 1 month.
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Figures

Videos

Literature Cited

Literature Cited
  Kinoshita‐Kikuta, E., Kinoshita, E., Matsuda, A., and Koike, T. 2014. Tips on improving the efficiency of electrotransfer of target proteins from Phos‐tag SDS‐PAGE gel. Proteomics 14:2437‐2442. doi: 10.1002/pmic.201400380.
  LeGendre, N. and Matsudaira, P. 1988. Direct protein microsequencing from Immobilon‐P transfer membrane. BioTechniques 6:154‐159.
  Mozdzanowski, J., Hembach, P., and Speicher, D. 1992. High yield electroblotting onto polyvinylidene difluoride membranes from polyacrylamide gels. Electrophoresis 13:59‐64. doi: 10.1002/elps.1150130112.
  Speicher, D.W. 1989. Microsequencing with PVDF membranes: Efficient electroblotting, direct protein adsorption and sequencer program modifications. In Techniques in Protein Chemistry (T. Hugli, ed.) pp. 24‐35, Academic Press, San Diego.
  Szewczyk, B. and Summers, D.F. 1988. Preparative elution of proteins blotted to Immobilon membranes. Anal. Biochem. 168:48‐53. doi: 10.1016/0003‐2697(88)90008‐5.
  Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76:4350‐4354. doi: 10.1073/pnas.76.9.4350.
  Vandekerckhove, J., Bauw, G., Puypi, M., Van Damme, J., and Van Montagu, M. 1985. Protein‐blotting on Polybrene‐coated glass‐fiber sheets. Eur. J. Biochem. 152:9‐19. doi: 10.1111/j.1432‐1033.1985.tb09157.x.
Key References
  LeGendre and Matsudaira, 1988. See above.
  Thoroughly reviews electroblotting using PVDF membranes.
  Mozdzanowski, J. and Speicher, D.W. 1992. Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types of PVDF membranes. Anal. Biochem. 207:11‐18.
  Compares electroblotting recoveries using different PVDF membranes.
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