Isolation of Proteins for Microsequence Analysis

Malcolm Moss1

1 Center for Biologics Evaluation & Research, Food and Drug Administration, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.19
DOI:  10.1002/0471142727.mb1019s21
Online Posting Date:  May, 2001
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Abstract

This unit presents a method for determining the amino‐terminal sequence of polypeptides or proteins. It is particularly appropriate for large fragments of insoluble or hydrophobic proteins or proteins that cannot be purified to >90% molar purity without electrophoresis. If the protein is blocked at the amino terminus, chemical cleavage or partial enzymatic digestion must be performed prior to electrophoresis. Upon isolation, the internal amino acid sequence is analyzed as described.

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Amino Acid Sequence by SDS‐PAGE and Transfer to PVDF Membranes
  • Support Protocol 1: Preparation of Protein Samples for SDS‐Page
  • Basic Protocol 2: Determination of Internal Amino Acid Sequence from Electrophoretically‐Separated Proteins
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Amino Acid Sequence by SDS‐PAGE and Transfer to PVDF Membranes

  Materials
  • Separating and stacking gel solutions (Table 10.19.1)
  • recipe4× gel buffer
  • Glutathione, reduced powder ( Sigma #G4251)
  • recipe10× lower reservoir buffer
  • recipe10× upper reservoir buffer
  • Mercaptoacetic acid, sodium salt
  • Protein sample in sample buffer (see protocol 2support protocol)
  • Methanol
  • recipeTransfer buffer
  • 0.1% Coomassie blue in 50% methanol (v/v)
  • 10% acetic acid in 50% methanol (v/v)
  • Vertical minigel unit (e.g., Bio‐Rad Mini‐Protean II; Hoefer Mighty Small SE 250/280 is not recommended for this procedure)
  • Power supply (constant voltage and constant current)
  • Microvolume syringe or gel‐loading pipet tip
  • Powder‐free plastic gloves
  • PVDF membranes (e.g., Immobilon‐P or ‐PSQ, Millipore; ProBlott, Applied Biosystems)
  • Small‐format transfer apparatus (Midget MultiBlot, Hoefer or Pharmacia LKB; Mini Trans‐Blot, Bio‐Rad)
  • Automated protein sequencer ( Applied Biosystems)
  • Additional reagents and equipment for minigel preparation (unit 10.2)
    Table 0.9.1   MaterialsRecipes for Polyacrylamide Separating and Stacking Gels

    SEPARATING GEL
    Stock solutions Final acrylamide concentration in the separating gels (%)
    5 6 7 8 9 10 11 12 13 14 15
    30% acrylamide monomer a 3.33 4.00 4.67 5.33 6.00 6.67 7.33 8.00 8.67 9.33 10.00
    H 2O 11.49 10.83 10.16 9.50 8.84 8.17 7.51 6.84 6.18 5.51 4.85
    TEMED b 0.040 0.033 0.029 0.025 0.025 0.020 0.018 0.017 0.015 0.014 0.013
     Mix the above ingredients (listed in milliliters of stock solution) with 5 ml of 4× gel buffer and 0.14 ml of 5% potassium persulfate or 70 µl of 10% ammonium persulfate.
    STACKING GEL
    • 0.666 ml 30% acrylamide monomer a3.033 ml H 2O0.025 ml 10% ammonium persulfate or 0.05 ml 5% potassium persulfate0.025 ml TEMED b

     aGas‐stabilized monomer solution (containing 37.5:1 acrylamide/N,N′‐methylene‐bisacrylamide) from which acrylic acid and carbonyl‐containing compounds have been removed (Protogel, National Diagnostics; or PAGE1 protein gel mix, Boehringer Mannheim)
     bTEMED may have to be altered to facilitate proper polymerization. Values given are reasonable approximations.

Support Protocol 1: Preparation of Protein Samples for SDS‐Page

  Additional Materials
  • Protein samples
  • 1 M NaHCO 3 (optional)
  • 100% ethanol, ice‐cold (containing no denaturants; USP grade)
  • recipeSample buffer
  • 0.1% (w/v) pyronin Y
  • Ultrafiltration concentrator ( Amicon) or Speedvac evaporator ( Savant)
  • Drawn‐out Pasteur pipet or gel‐loading pipet tip
  • Boiling water bath

Basic Protocol 2: Determination of Internal Amino Acid Sequence from Electrophoretically‐Separated Proteins

  Materials
  • Protein sample
  • 0.1% (w/v) Ponceau S ( Sigma) in 1% (v/v) acetic acid
  • 1% acetic acid (v/v)
  • 0.2 mM NaOH
  • 0.5% (w/v) polyvinylpyrrolidone ( Sigma # PVP‐40) in 0.1 M acetic acid
  • recipeDigestion buffer
  • 1 mg/ml sequencing‐grade trypsin ( Promega #V511A)
  • Chromatography solvent A: 5% (v/v) acetonitrile in 0.1% (v/v) trifluoracetic acid (TFA)
  • Chromatography solvent B: 70% (v/v) acetonitrile in 0.085% (v/v) TFA
  • 0.22–µm nitrocellulose membrane (e.g., Schleicher & Schuell #BA83)
  • Acid‐washed glass plate or petri dish
  • Powder‐free gloves
  • Fine‐tipped forceps
  • 0.5‐ml microcentrifuge tube
  • Bath sonicator (e.g., Bransonic 12)
  • Centrifugal filter device, 0.22‐µm membrane, low‐protein‐binding (e.g., Millipore #UFC3‐OGV‐00)
  • Reversed‐phase HPLC column (e.g., Vydac #214TP52), UV column monitor, and chart recorder
  • Column oven (optional)
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Figures

Videos

Literature Cited

Literature Cited
   Aebersold, R.H., Teplow, D.B., Hood, L.E., and Kent, S.B.H. 1986. Electroblotting onto activated glass. J. Biol. Chem. 261:4229‐4238.
   Aebersold, R.H., Leavitt, J., Saavedra, R.A., Hood, L.E., and Kent, S.B.H. 1987. Internal amino acid sequence analysis of proteins separated by one ‐ or two ‐ dimensional electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci. U.S.A. 84:6970‐6974.
   Hewick, R.M., Hunkapiller, M.W., Hood, L.E., and Dreyer, W.J. 1981. A gas‐ liquid solid phase peptide and protein sequenator. J. Biol. Chem. 256:7990‐7997.
   Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:10035‐10038.
   Moos, M., Nguyen, N.Y., and Liu, T.‐Y. 1988. Reproducible, high‐ yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem. 263:6005‐6008.
   Schägger, H. and von Jagow, G. 1987. Tricine‐sodium dodecylsulfate‐polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa. Anal. Biochem. 166:368‐379.
   Sheer, D.G., Yuen, S.W., Yamone, D.K., Mattaliano, R.J., and Yuan, P.‐M. 1990. A modified reaction cartridge for sequencing samples on polymeric sample matrices. Abstract T139, Fourth Symposium of the Protein Society San Diego.
   Speicher, D.W. 1989. Microsequencing with PVDF membranes: Efficient electroblot‐ting, direct protein adsorption and sequencer program modifications. In Techniques in Protein Chemistry (T.E. Hugli, ed.) pp. 24‐35. Academic Press, San Diego.
   Stone, K.L. and Williams, K.R. 1986. High‐ performance liquid chromatographic peptide mapping and amino acid analysis in the subnanomole range. J. Chromatogr. 359:203‐212.
   Tempst, P. and Riviere, L. 1989. Examination of automated polypeptide sequencing using standard phenylisothiocyanate reagent and subpicomole high‐ performance liquid chromatographic analysis. Anal. Biochem. 183:290‐300.
   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 picomole level: Improved methods, applications and gene cloning strategies. Electrophoresis 11:537‐553.
   Vandekerckhove, J., Bauw, G., Puype, M., van Damme, J., and van Montagu, M. 1986. Protein blotting from polyacrylamide gels onto glass microfiber filters. In Advanced Methods in Protein Microsequence Analysis (B. Wittmann‐Liebold, J. Salnikow, and V.A. Erdman, eds.) pp. 179‐193. Springer‐Verlag, Berlin.
   Yuan, P., Hawke, D., Blacher, R., Hunkapiller, M., and Wilson, K. 1987. Ethanol precipitation of electroeluted‐ electrodialyzed samples. Applied Biosystems User Bulletin #27.
Key References
   Moos et al., 1988. See above.
  Address common problems encountered in procedures of this type.
   Tempst, et al. See above.
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