Enzymatic Digestion of Proteins on PVDF Membranes

Joseph Fernandez1, Sheenah M. Mische1

1 The Rockefeller University, New York, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.2
DOI:  10.1002/0471140864.ps1102s00
Online Posting Date:  May, 2001
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Abstract

Enzymatic digestion of membrane‐bound proteins is one of the most widely used procedures for determining the internal amino acid sequence of proteins that either have a blocked amino terminus or require two or more stretches of sequence data for DNA cloning or confirmation of protein identification. Because the final step of protein purification is usually SDS‐PAGE, electroblotting to either polyvinylidene difluoride (PVDF) or nitrocellulose is the simplest and most common procedure for recovering protein free of contaminants (e.g., SDS or acrylamide) with a high yield. As described in this unit, PVDF is preferred over nitrocellulose because it can be used for a variety of other structural analysis procedures, such as amino‐terminal sequence analysis and amino acid analysis. In addition, peptide recovery from PVDF membranes is higher than from nitrocellulose, particularly from higher‐retention PVDF (e.g., ProBlott, Transblot, Westran, or Immobilon Psp). Finally, PVDF‐bound protein can be stored dry, as opposed to nitrocellulose‐bound protein, which must remain wet during handling and storage to prevent loss of peptides during digestion.

     
 
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Table of Contents

  • Basic Protocol 1: Digestion of PVDF‐Bound Proteins in a Hydrogenated Triton X‐100 Buffer
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Digestion of PVDF‐Bound Proteins in a Hydrogenated Triton X‐100 Buffer

  Materials
  • Protein samples electrophoresed on SDS‐polyacrylamide gel (units 10.1 10.4)
  • PVDF membrane (e.g., ProBlott, Immobilon Psq, or Westran)
  • Milli‐Q water or equivalent
  • recipeDigestion buffer (see recipe and Table 11.2.1)
  • Appropriate 0.1 µg/µl recipeenzyme solution (depending on the cleavage site required; see recipe)
  • 0.1% (v/v) trifluoroacetic acid (TFA) in Milli‐Q water or equivalent
  • recipe1% (v/v) diisopropylfluorophosphate (DFP) in ethanol (see recipe)
  • Powder‐free gloves
  • Glass plate
  • Forceps
  • Sonicating water bath
  • Injection vials for HPLC
  • Microbore HPLC
  • Clean capless 1.5‐ml microcentrifuge tubes and separate caps (e.g., Sarstedt)
  • Additional reagents and equipment for electroblotting (unit 10.7), staining transferred proteins (unit 10.8), and reversed‐phase HPLC (unit 11.6)
    Table 1.2.1   Materials   Digestion Buffers for Various Enzymes a   Digestion Buffers for Various Enzymes

    Enzyme Digestion buffer Recipe Comments
    Trypsin or endoproteinase Lys‐C 1% RTX‐100/10% acetonitrile/100 mM Tris⋅Cl, pH 8.0 100 µl 10% RTX‐100 stock 100 µl acetonitrile 300 µl HPLC‐grade water 500 µl 200 mM Tris⋅Cl, pH 8.0 RTX‐100 prevents enzyme adsorption to membrane and increases recovery of peptides
    Endoproteinase Glu‐C 1% RTX‐100/100 mM Tris⋅Cl, pH 8.0 100 µl 10% RTX‐100 stock 400 µl HPLC‐grade water 500 µl 200 mM Tris⋅Cl, pH 8.0 Acetonitrile is omitted because it decreases digestion efficiency of endoproteinase Glu‐C
    Clostripain 1% RTX‐100/10% acetonitrile/2 mM DTT/1 mM CaCl 2/100 mM Tris⋅Cl, pH 8.0 100 µl 10% RTX‐100 stock 100 µl acetonitrile 45 µl 45 mM DTT 10 µl 100 mM CaCl 2 245 µl HPLC‐grade water 500 µl 200 mM Tris⋅Cl, pH 8.0 DTT and CaCl 2 are necessary for clostripain activity

     aSee appendix 2E for Tris⋅Cl and DTT stock solution recipes. Abbreviations: DTT, dithiothreitol; RTX‐100, hydrogenated Triton X‐100 (Tiller et al., ).
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Figures

Videos

Literature Cited

Literature Cited
   Aebersold, R. 1993. Internal amino acid sequence analysis of proteins after in situ. protease digestion on nitrocellulose. In A Practical Guide to Protein and Peptide Purification for Microsequencing, 2nd Ed. (P. Matsudaira, ed.) pp. 105‐154. Academic Press, New York.
   Aebersold, R.H., Leavitt, J., Saavedra, R.A., Hood, L.E., and Kent, S.B. 1987. Internal amino acid sequence analysis of proteins separated by one‐ or two‐dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci. U.S.A. 84:6970‐6974.
   Atherton, D. 1989. Successful PTC amino acid analysis at the picomole level. In Techniques in Protein Chemistry (T. Hugli, ed.) pp. 273‐283. Academic Press, New York.
   Bauw, G., Van Damme, J., Puype, M., Vandekerckhove, J., Gesser, B., Ratz, G.P., Lauridsen, J.B., and Celis, J.E. 1989. Protein‐electroblotting and ‐microsequencing strategies in generating protein data bases from two‐dimensional gels. Proc. Natl. Acad. Sci. U.S.A. 86:7701‐7705.
   Best, S., Reim, D.F., Mozdzanowski, J., and Speicher, D.W. 1994. High sensitivity sequence analysis using in situ proteolysis on high retention PVDF membranes and a biphasic reaction column sequencer. In Techniques in Protein Chemistry V (J. Crabb, ed.) pp. 205‐213. Academic Press, New York.
   Fernandez, J., DeMott, M., Atherton, D., and Mische, S.M. 1992. Internal protein sequence analysis: Enzymatic digestion for less than 10 µg of protein bound to polyvinylidene difluoride or nitrocellulose membranes. Anal. Biochem. 201:255‐264.
   Fernandez, J., Andrews, L., and Mische, S.M. 1994a. An improved procedure for enzymatic digestion of polyvinylidene difluoride‐bound proteins for internal sequence analysis. Anal. Biochem. 218:112‐118.
   Fernandez, J., Andrews, L., and Mische, S.M. 1994b. A one‐step enzymatic digestion procedure for PVDF‐bound proteins that does not require PVP‐40. In Techniques in Protein Chemistry V (J. Crabb, ed.) pp. 215‐222. Academic Press, New York.
   Mozdzanowski, J. and Speicher, D.W. 1990. Quantitative electrotransfer of proteins from polyacrylamide gels onto PVDF membranes. In Current Research in Protein Chemistry: Techniques, Structure, and Function. (J. Villafranca, ed.) pp. 87‐94. Academic Press New York.
   Tempst, P., Link, A.J., Riviere, L.R., Fleming, M., and Elicone, C. 1990. Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: Improved methods, applications and gene cloning strategies. Electrophoresis. 11:537‐553.
   Tiller, G.E., Mueller, T.J., Dockter, M.E., and Struve, W.G. 1984. Hydrogenation of Triton X‐100 eliminates its fluorescence and ultraviolet light absorbance while preserving its detergent properties. Anal. Biochem. 141:262‐266.
Key References
   Fernandez et al., 1994a. See above.
  Describes digestion with and without PVP‐40 and applies it to unknown proteins.
   Fernandez et al., 1994b. See above.
  Describes digestion procedure and emphasizes applicability to different types of PVDF membranes and the concentration of RTX‐100 buffer.
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