Removal of Detergents from Proteins and Peptides in a Spin‐Column Format

Babu S. Antharavally1

1 Thermo Fisher Scientific, Research & Development, Rockford, Illinois
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.12
DOI:  10.1002/0471140864.ps0612s69
Online Posting Date:  August, 2012
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Abstract

To enable downstream analysis, it is critical to remove unbound detergents from protein and peptide samples. This unit describes the use of a high‐performance resin that offers exceptional detergent removal for proteins and peptides. The easy‐to‐use spin format significantly improves results over the standard drip column and batch methodologies, with >95% removal of 1% to 5% detergents, including SDS, sodium deoxycholate, CHAPS, Triton X‐100, Triton X‐114, NP‐40, Brij‐35, octyl glucoside, octyl thioglucoside, and lauryl maltoside, with high recovery of proteins and peptides. Detergent removal efficiency is evaluated using colorimetric methods and mass spectrometry (MS). BSA tryptic peptides have been successfully analyzed by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) and matrix‐assisted laser desorption/ionization (MALDI)‐MS for identification of protein, following detergent removal using the resin. Advantages of this method include speed (less than 15 min), efficient detergent removal, and high recovery of proteins and peptides. Curr. Protoc. Protein Sci. 69:6.12.1‐6.12.7. © 2012 by John Wiley & Sons, Inc.

Keywords: detergent removal; spin‐column format; proteins and peptides; downstream analysis; mass spectrometry

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • 50 mM ammonium bicarbonate buffer, pH 8.0 (see recipe)
  • Protein sample containing detergent
  • 1.5‐ and 2‐ml microcentrifuge collection tubes
  • Variable‐speed benchtop microcentrifuge
  • Additional reagents and equipment for BCA (bicinchoninic acid) protein assay method [unit 3.1or or as required for BCA protein assay kit (Thermo Fisher Scientific)], and for detection of detergent in sample (see Table 6.12.1 and Chapter 16)
CAUTION: The spin columns contain sodium azide, which is toxic. Wear gloves and follow all precautions supplied by the manufacturer for this chemical.
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Figures

Videos

Literature Cited

Literature Cited
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   Bollag, D.M. and Edelstein, S.J. 1991. Protein Methods. Wiley‐Liss, New York.
   Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X‐114 solution. J. Biol. Chem. 256:1604‐1607.
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   Cohen, S.L. and Chait, B.T. 1996. Influence of matrix solution conditions on the MALDI‐MS analysis of peptides and proteins. Anal. Chem. 68:31‐37.
   Funk, J., Li, X., and Franz, T. 2005. Threshold values for values for detergents in protein and peptide samples for mass spectrometry. Rapid Commun. Mass Spectrom. 19:2986‐2988.
   Furth, A.J., Bolton, H., Potter, J., and Priddle, J.D. 1984. Separating detergents from proteins. Methods Enzymol. 104:318‐328.
   Hjelmeland, L.M. 1990. Removal of detergents from membrane proteins. Methods Enzymol. 182:277‐282.
   Loo, R.R.O., Dales, N., and Andrews, P.C. 1996. The effect of detergents on proteins analyzed by electrospray ionization. Methods Mol. Biol. 61:141‐160.
   Makino, S., Woolford, J.L. Jr., Tanford, C., and Webster, R.E. 1975. Interaction of deoxycholate and of detergents with the coat protein of bacteriophage F1. J. Biol. Chem. 250:4327‐4332.
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