Protein Biotinylation

Giuliano Elia1

1 University College Dublin, Dublin, Ireland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 3.6
DOI:  10.1002/0471140864.ps0306s60
Online Posting Date:  April, 2010
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Abstract

Since its discovery in the first half of the twentieth century, the high‐affinity, noncovalent interaction between biotin (vitamin H) and the avian protein avidin (and its bacterial homologs) has been exploited for many diverse biotechnology applications. This unit provides several basic protocols for labeling various protein reactive groups with biotin. These protocols can be applied not only to labeling in vitro or in tissue culture, but also to in vivo labeling of whole laboratory animals or to ex vivo labeling of surgically resected organs. Curr. Protoc. Protein Sci. 60:3.6.1‐3.6.21. © 2010 by John Wiley & Sons, Inc.

Keywords: biotinylation; cell surface proteins; in vivo biotinylation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Biotinylation of a Purified Protein in Solution: Modification of Free Primary Amino Groups
  • Alternate Protocol 1: Biotinylation of a Purified Protein in Solution: Modification of Free Thiol Groups
  • Alternate Protocol 2: Biotinylation of a Purified Protein in Solution: Modification of Carbohydrates in Glycans
  • Alternate Protocol 3: Biotinylation of a Purified Protein in Solution: Use of Photobiotin
  • Basic Protocol 2: Cell Surface Biotinylation
  • Basic Protocol 3: In Vivo Biotinylation of Mice Through Intra‐Cardiac Perfusion
  • Support Protocol 1: Capture and Release of Biotinylated Proteins from Streptavidin‐Sepharose
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Biotinylation of a Purified Protein in Solution: Modification of Free Primary Amino Groups

  Materials
  • Protein mixture or purified protein of interest: e.g., bovine serum albumin (BSA), mol. wt., ∼66,000 (Sigma, cat. no. A‐7030)
  • Appropriate buffer containing no free primary amino groups: e.g., 50 mM phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • Sulfosuccinimidyl‐6‐(biotinamido)hexanoate (Sulfo‐NHS‐LC‐biotin, mol. wt. 556.59; e.g., Thermo Scientific, cat. no., 21335)
  • Buffer containing primary amino groups for quenching: e.g., 50 mM Tris⋅Cl, pH 7.4 ( appendix 2E), in Milli‐Q water
  • PD‐10 desalting columns (GE Healthcare, cat. no. 17‐0851‐01; optional)
  • Vivaspin concentrators (VS 20; MWCO, 10,000 Da; volume, 20 ml; Vivascience AG, http://www.vivascience.com)
  • Additional reagents and equipment for determining protein concentration (unit 3.4)

Alternate Protocol 1: Biotinylation of a Purified Protein in Solution: Modification of Free Thiol Groups

  Materials
  • Protein mixture or purified protein of interest (see protocol 1)
  • 50 mM phosphate‐buffered saline (PBS), pH 6.5 to 7.5 (see recipe), containing 10 mM EDTA
  • 1‐biotinamido‐4‐[4′‐(maleimidomethyl)‐cyclohexyl carboxamido]butane (Biotin‐BMCC, mol. wt., 533.69; e.g., Thermo Scientific, cat. no. 21900)
  • Dimethylsulfoxide (DMSO)
  • Additional reagents and equipment for purifying biotinylated proteins by size‐exclusion chromatography (see protocol 1)

Alternate Protocol 2: Biotinylation of a Purified Protein in Solution: Modification of Carbohydrates in Glycans

  Materials
  • Protein mixture or purified protein of interest (see protocol 1)
  • 50 mM sodium phosphate buffer, pH 7.0 ( appendix 2E), prepared with Milli‐Q water
  • Sodium metaperiodate (mol. wt., 213.91; Sigma, cat. no. S‐1878)
  • Biotin‐LC‐hydrazide (mol. wt. 371.50; e.g., Thermo Scientific, cat. no. 21340)
  • Dimethylsulfoxide (DMSO)
  • PD‐10 desalting columns (GE Healthcare; Cat# 17‐0851‐01)
  • Vivaspin concentrators VS 20 (MW cut‐off 10,000 Da, vol = 20 ml) (Vivascience AG)
  • Additional reagents and equipment for size‐exclusion chromatography with PD‐10 columns and buffer exchange/concentration with Vivaspin centrifugal concentrators ( protocol 1)

Alternate Protocol 3: Biotinylation of a Purified Protein in Solution: Use of Photobiotin

  Materials
  • Protein mixture or purified protein of interest (see protocol 1)
  • 50 mM phosphate‐buffered saline (PBS), pH 7.0 (see recipe)
  • Tetrafluorophenylazide‐(triethyleneglycol)‐Biotin (TFPA‐PEG 3‐Biotin; mol. wt., 663.69; e.g., Thermo Scientific, cat. no. 21303)
  • Dimethylsulfoxide (DMSO) or dimethylformamide (DMF)
  • Amber (or foil‐covered) microcentrifuge tubes
  • Shallow, low‐protein‐binding vessel for irradiation, e.g., Thermo Nunc HydroCell 10‐cm dishes (cat. no. 174911)
  • UV lamp: e.g., Stratalinker 2400 (Stratagene) or Spectroline (Spectronics)
  • PD‐10 desalting columns (GE Healthcare; Cat# 17‐0851‐01)
  • Additional reagents and equipment for size‐exclusion chromatography (see protocol 1)

Basic Protocol 2: Cell Surface Biotinylation

  Materials
  • Cells growing in 75‐cm2 tissue culture flasks and appropriate growth medium
  • 50 mM phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • Sulfosuccinimidyl‐2‐(biotinamido)ethyl‐1,3‐dithiopropionate (Sulfo‐NHS‐SS‐biotin, mol. wt., 606.7; e.g., Thermo Scientific, cat. no. 21331)
  • Buffer containing primary amino groups for quenching: e.g., 50 mM Tris⋅Cl, pH 7.4 ( appendix 2E)
  • 1 mM oxidized glutathione (mol. wt., 612.6; e.g., Sigma, cat. no. G‐4376) dissolved in 50 mM PBS (see recipe)
  • Lysis buffer of choice, e.g., if protocol 7 is the downstream application, see recipe for lysis buffer in Reagents and Solutions
  • Cell scrapers
  • Centrifuge

Basic Protocol 3: In Vivo Biotinylation of Mice Through Intra‐Cardiac Perfusion

  Materials
  • Mouse or mice to be perfused
  • 50 mM phosphate‐buffered saline (PBS), pH 7.4 (see recipe), supplemented with 10% (w/v) dextran 40 (e.g., Pharmacosmos, cat. no. 5510 0040 1006; http://www.pharmacosmos.com/)
  • Biotinylation solution: 1 mg/ml sulfo‐NHS‐LC‐biotin (e.g., Thermo Scientific, cat. no. 21335) in 50 mM PBS (pH 7.4)/10% (w/v) dextran 40
  • Ketamine (e.g., Sigma, cat. no. K‐2753)
  • Xylazine (e.g., Sigma, cat. no. X‐1251)
  • Physiological saline (0.9% w/v NaCl), sterile
  • Acepromazine (e.g., Boehringer‐Ingelheim, cat. no. 670 025)
  • Quenching solution: 50 mM Tris⋅Cl in 50 mM PBS (pH 7.4)/10% (w/v) dextran 40
  • Liquid N 2
  • Isopentane
  • OCT cryoembedding compound (Tissue Tek or Sakura Finetek)
  • Lysis buffer (see recipe)
  • Perfusion device (Fig. ) comprising:
    • 25‐G butterfly cannula with barbed point (e.g., Vita Needle Company, http://www.vitaneedle.com/)
    • Three‐way T‐split valves
    • Manometer (e.g., Bosch Gmbh; http://aa.bosch.de)
    • Peristaltic pump
    • Reservoirs for perfusion solutions
    • Infrared lamp
  • Surgical instruments including precision scissors
  • Homogenizer (e.g., T8 Ultra‐Turrax; IKA Labortechnik Cat. # 8002000)
  • Heating block (e.g., Thermomixer Comfort, Eppendorf)
  • Centrifuge
  • Additional reagents and equipment for cryosectioning (Watkins, )

Support Protocol 1: Capture and Release of Biotinylated Proteins from Streptavidin‐Sepharose

  Materials
  • BioRad Protein Assay kit (BioRad, cat. no. 500‐0002; also see unit 3.4)
  • Biotinylated BSA ( protocol 1)
  • 50 mM phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • Streptavidin‐Sepharose High Performance (GE Healthcare, cat. no. 17‐5113‐01), prewashed (see recipe)
  • Wash buffer A (see recipe)
  • Wash buffer B (see recipe)
  • Elution solution (see recipe)
  • Revolving mixer (e.g., Reax‐2 from Heidolph Instruments)
  • Ultrafree‐MC centrifugal filters (5.0 µm Durapore filter units; Millipore, cat. no. UFC30SV00)
  • Heating block (e.g., Thermomixer Comfort, Eppendorf)
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Figures

  •   FigureFigure 3.6.1 Schematic design of a reagent for biotin labeling of proteins. All reagents contain at one end the bicyclic ring of biotin (or iminobiotin) and the valeric acid side chain, to which a linear spacer group of different length and chemical composition may be attached through formation of an amide bond. At the opposite end of the compound sits the chemical group responsible for carrying out the covalent binding of the biotin‐linker moiety to suitable chemical groups in proteins. A few examples of commercially available spacer arms (R1) and of reactive groups (R2) are shown.
  •   FigureFigure 3.6.2 Chemical structure and size of some common N‐hydroxysuccinimido esters of biotin.
  •   FigureFigure 3.6.3 Biotinylating reagents for protein derivatization. Footnotes: a, ɛ‐amino groups in side chains of lysines and unsubstituted protein N‐terminal α‐amino groups; b, same as a as well as secondary amino groups in arginine and histidine; c, reduced sulfhydryl groups in cysteines; d, aldehyde groups can be created by periodate oxidation of carbohydrate residue. Chemical abbreviations: BMCC, 1‐biotinamido‐4‐[4′‐(maleimidoethyl‐cyclohexane)‐carboxamido]butane; MAL, maleimido; NHS, N‐hydroxysuccimide; LC, long‐chain; PEO, polyethylene oxide; PFP, pentafluorophenyl; TFP, tetrafluorophenyl. Supplier abbreviations: CP, ChemPep (http://www.chempep.com); F, Fluka; K, Kirkegaard & Perry Laboratories; PE, Perkin‐Elmer; Q, Quanta Biodesign; T, Thermo.
  •   FigureFigure 3.6.4 Structure of photobiotin and its chemical reaction. Upon irradiation with UV light at a wavelength of 320 nm, the aryl azide is photolyzed to form an aryl‐nitrene group. Derivatization can then occur at reactive hydrogen sites or at double bonds or, as in the illustrated example, upon ring expansion of the nitrene group to form a dehydroazepine derivative, which reacts with nucleophilic groups (e.g., primary amines in proteins).
  •   FigureFigure 3.6.5 Chemical structure and size of sulfosuccinimidyl‐2‐(biotinamido) ethyl‐1,3‐dithiopropionate (Sulfo‐NHS‐SS‐biotin).
  •   FigureFigure 3.6.6 Schematic diagram of the experimental setup for the intra‐cardiac perfusion of mice with biotinylating reagents.

Videos

Literature Cited

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