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In vitro enzyme assay of nucleocytoplasmic O-linked β-N-acetylglucosaminyltransferase (OGT)
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In vitro enzyme assay of nucleocytoplasmic O-linked β-N-acetylglucosaminyltransferase (OGT)

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Introduction Protocol References Credit lines
Category
Glycosyltransferases & related proteins
Protocol Name

In vitro enzyme assay of nucleocytoplasmic O-linked β-N-acetylglucosaminyltransferase (OGT)

Authors
 Kamemura, Kazuo
Department of Bioscience, Nagahama Institute of Bio-Science & Technology
KeyWords
OGT   O-GlcNAc  
Reagents
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A synthetic peptide (PGGSTPVSSANMM-CK II peptide or an artificial peptide YSDSPSTST)

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UDP-[6-3H]GlcNAc (0.74–1.66 TBq/mmol (3.7 MBq/mL): PerkinElmer, Waltham, MA)

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5’-Adenosine monophosphate (5’-AMP)

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Bovine serum albumin (BSA)

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Sodium fluoride (if necessary)

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1-Amino-GlcNAc (if necessary)

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SP-Sephadex (SP-C25-120: Sigma-Aldrich, St. Louis, MO) or C18 cartridge (Sep-Pak: Waters Corp., Milford, MA).

Instruments
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Liquid scintillation counter
Methods
1.

In vitro enzyme assay of nucleocytoplasmic O-linked β-N-acetylglucosaminyltransferase (OGT)

1) 

 Prepare OGT enzyme fraction.

 Comment 1
2) 

 Prepare the reaction mixture (final volume of 50 μL).

  • 50 mM sodium cacodylate (pH 6.0–7.0)
  • 5 mM MnCl2
  • 3-15 mM synthetic peptide
  • 1.85-3.7 kBq of UDP-[6-3H]GlcNAc
  • 2.5 mM 5’-AMP
  • 1 mg/mL BSA
  • OGT enzyme fraction
 Comment 1
3) 

 Incubate at 20ºC for 30–60 min.

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4) 

 Stop the reaction by the addition of 450 μL of 50 mM formic acid.

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5) 

 Separate the labeled peptide from unincorporated label by SP-Sephadex or C18 cartridge.

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If you use SP-Sephadex:

Load the reaction mixture directly onto a 0.5-mL SP-Sephadex column equilibrated with 50 mM formic acid.

↓

Wash with 10 mL of 50 mM formic acid.

↓

Elute the labeled peptide with 1 mL of 0.5 M NaCl.

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If you use C18 cartridge:

Load the reaction mixture directly onto a C18 cartridge equilibrated with 50 mM formic acid.

↓

Wash sequentially with 10 mL of 50 mM formic acid, 10 mL of 50 mM formic acid containing 1 M NaCl, and 10 mL of MilliQ water.

↓

Elute the labeled peptide with 5 mL of 50% methanol.

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6) 

 Measure the incorporation of the [3H]GlcNAc label on the synthetic peptide by liquid scintillation counting.

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Figure & Legends

Figure & Legends

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Date of registration：2014-06-04 10:11:44
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