Detection of Tyrosine Sulfation on Proteins

Yogita Kanan1, Muayyad R. Al Ubaidi1

1 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.7
DOI:  10.1002/0471140864.ps1407s80
Online Posting Date:  April, 2015
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Abstract

Tyrosine sulfation is a post‐translational modification (PTM) where a sulfate group is added to a tyrosine moiety. This PTM is responsible for strengthening interaction between proteins. One of the drawbacks of studying this PTM is the lack of an antibody that can detect all tyrosine‐sulfated proteins. In addition, due to the labile nature of the tyrosine sulfate, other techniques such as mass spectrometry cannot be used to study this PTM unless special modification procedures are used. This requires considerable skill and knowledge of mass spectrometry. This unit describes an in vitro technique that can be used to study tyrosine‐sulfated proteins by radiolabeling the recombinant protein. The protein is then subject to barium hydroxide hydrolysis and thin‐layer electrophoresis (TLE). Co‐localization of radioactive tyrosine‐sulfate with nonradioactive tyrosine sulfate standard spiked in before TLE analysis identifies a protein as tyrosine‐sulfated protein. The advantage of this technique is that, it identifies all tyrosine‐sulfated proteins without any bias and is the only technique that identifies the tyrosine sulfate residues in the protein. © 2015 by John Wiley & Sons, Inc.

Keywords: tyrosine sulfate; sulfotyrosine; post‐translational modification; thin‐layer electrophoresis; barium hydroxide hydrolysis; autoradiography

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Metabolic Labeling of Protein of Interest with 35S Sulfate In Vitro and Immunoprecipitation
  • Support Protocol 1: Detection of Radioactive Tyrosine‐Sulfated Protein by Autoradiography
  • Reagents and Solutions
  • Commentary
  • Literature cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Metabolic Labeling of Protein of Interest with 35S Sulfate In Vitro and Immunoprecipitation

  Materials
  • Fetal bovine serum (FBS; Life Technologies)
  • Penicillin‐streptomycin, 100× (Sigma Aldrich)
  • Dulbecco's modified Eagle medium (DMEM; Life Technologies)
  • Human embryonic kidney epithelial cells 293 T (ATCC)
  • 1× Dulbecco's phosphate‐buffered saline (D‐PBS; Life Technologies)
  • Cell dissociation reagent TrypLE (Life Technologies)
  • 0.5 μg/ml recombinant clone of gene of interest (Genecopoeia, Life Technologies, Origene or similar source)
  • 2 M CaCl 2 (see recipe)
  • 1× Tris‐EDTA solution (see recipe)
  • 2× HBS solution (see recipe)
  • Dialyzed fetal bovine serum (FBS; Sigma Aldrich)
  • Joklik's modified Eagle medium (JMEM; Sigma Aldrich), 500 ml
  • 5 mCi radioactive sodium sulfate (Na35SO 4; PerkinElmer, cat. no. NEX041H)
  • Protein A/G agarose (Thermo Scientific)
  • Primary antibody against recombinant clone (any commercial antibody source)
  • Isotype control antibodies raised against primary antibody's host species (any commercial source)
  • 2× SDS sample buffer (see recipe)
  • 1× Dulbecco's phosphate‐buffered saline containing 0.1% (v/v) Tween 20 (D‐PBS‐T; Life Technologies)
  • Ice
  • Distilled water
  • BSA powder (Sigma)
  • Bradford reagent (Bio‐Rad)
  • 10‐cm tissue culture dishes (Corning)
  • 1‐, 5‐, and 25‐ml sterile pipets (Corning)
  • 5% CO 2 tissue culture incubator
  • Tissue culture laminar flow hood
  • Hemacytometer, optional
  • 6‐well tissue culture plate (Corning), optional
  • 1.5‐ml microcentrifuge tubes
  • Laboratory centrifuge
  • 15‐ml Falcon conical centrifuge tubes (Fischer Scientific)
  • Vortex mixer
  • Nutator mixer
  • 100°C heating block
  • Cell scraper (Corning)
  • Sonicator
  • UV‐visible Spectrometer
CAUTION: Radioactive materials require special handling. See appendix 2B concerning safe use of radioisotopes.

Support Protocol 1: Detection of Radioactive Tyrosine‐Sulfated Protein by Autoradiography

  Additional Materials (also see protocol 1Basic Protocol)
  • Immunoprecipitated sample (see the protocol 1Basic Protocol)
  • Prestained protein markers
  • 10% SDS Polyacrylamide gels
  • PVDF membrane (Millipore)
  • Distilled water
  • Barium hydroxide (Fisher Scientific)
  • Degassed water
  • Methanol
  • 10% phenol red solution
  • 1 M sulfuric acid
  • EB buffer (see recipe)
  • 1% Ninhydrin solution in glacial acetic acid
  • Tyrosine sulfate and serine sulfate standards (Bachem): 2 mg/ml stock of each
  • Gel electrophoresis apparatus
  • X‐ray cassette with intensifying screen
  • Phosphorescent marking pen (Electron Microscopy Services)
  • Plastic wrap
  • X‐ray film (Kodak)
  • Freezer (−80°C)
  • Table top light box
  • Glass tubes with screw caps
  • Heating block
  • Centrifuge
  • 2‐ml microcentrifuge tubes
  • Speed vacuum concentrator
  • Whatman 3MM filter paper
  • Gel‐loading tips attached to low‐volume pipets
  • 1‐ml syringes attached to 0.22‐μm filter
  • Hunter's Thin Layer Electrophoresis System (HTLE)
  • Fume hood
  • Hot air drier
  • Aerosol sprayer
  • TLC cellulose sheet 20 × 20 cm (EMD Chemicals)
  • Plastic sheet protector
  • Document scanner
CAUTION: All supplies in contact with the gel and membrane contain radioactivity. Use special caution in downstream applications.
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Figures

Videos

Literature Cited

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