Protein Truncation Test

Rolf Vossen1, Johan T. den Dunnen1

1 Human and Clinical Genetics, Leiden University Medical Center, Leiden
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.11
DOI:  10.1002/0471142905.hg0911s42
Online Posting Date:  September, 2004
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Abstract

The protein truncation test detects mutations at the protein level that lead to premature translation termination. The method has evolved considerably since is original publication in this manual. This thoroughly revised unit describes what is now the preferred method for performing the protein truncation test. Transcription and translation are performed in separate reactions; during translation, biotin‐labeled or N‐terminally tagged proteins are synthesized. The translation products are detected on immunoblots via chemiluminescence. An Alternate Protocol using coupled in vitro transcription/translation and radiolabeled proteins is also presented.

     
 
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Table of Contents

  • Basic Protocol 1: Nonradioactive Protein Truncation Test
  • Support Protocol 1: Isolation and Analysis of RNA
  • Alternate Protocol 1: Radioactive Protein Truncation Test Using Coupled Transcription/Translation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Nonradioactive Protein Truncation Test

  Materials
  • 1 to 3 µg patient and control RNA in ethanol (see protocol 2) or DNA sample
  • 3 M sodium acetate, pH 5.6 ( appendix 2D; pH adjusted to 5.6 with acetic acid)
  • 100% ethanol
  • Expand Reverse Transcriptase kit (Roche Diagnostics) containing 50 U/µl Expand reverse transcriptase and 5× buffer
  • 100 mM dithiothreitol (DTT; e.g., Life Technologies)
  • 0.5 µg/µl random primer (e.g., Promega)
  • 10 mM 4dNTP mix ( appendix 2D)
  • 40 U/µl ribonuclease inhibitor (e.g., RNasin, Promega)
  • 1.5 U/µl RNase H (e.g., Promega)
  • 10 mg/ml BSA (e.g, Roche Diagnostics)
  • 10× PCR buffer (e.g., SuperTaq buffer, HT Biotechnologies; or appendix 2D)
  • 5 U/µl Taq DNA polymerase (Amplitaq, Perkin Elmer)
  • Expand High Fidelity PCR System (Roche Diagnostics) containing:
    • 3.5 U/µL enzyme mix
    • 10× Expand HF buffer with and without 15 mM MgCl 2
    • 2.5 M MgCl 2
  • 20 pmol/µl gene‐specific primers for PCR: forward, internal tailed forward, reverse, and internal reverse primers (see and Fig. )
  • Mineral oil (e.g., Light Oil, Sigma)
  • Nonradioactive Protein Truncation Test kit (Roche Diagnostics) containing:
    • Control DNA (200 ng/µl human genomic DNA)
    • 500 ng/µl 5′ control primer
    • 150 ng/µl 3′ control primer
    • 4× transcription mix
    • T7 translation mix (including biotin‐labeled lysine, if used)
    • 25 mM magnesium acetate solution
    • 25 mM sodium EDTA solution
    • Sterile RNAse‐free water
    • Combi‐marker (biotin + color)
    • SDS sample buffer
  • 30% (w/v) 37.5:1 acrylamide/bisacrylamide (see recipe)
  • Tris⋅Cl, pH 6.6 and pH 8.8 ( appendix 2D)
  • 10% SDS ( appendix 2D)
  • 10% (w/v) ammonium persulfate (APS; freshly prepared)
  • N,N,N′,N′‐Tetramethylethylenediamine (TEMED)
  • Running buffer (see recipe)
  • Dimethylsulfoxide (DMSO)
  • PVDF Western blotting membrane (Roche Diagnostics) or equivalent
  • Blotting buffer (see recipe), ice cold
  • Tris‐buffered saline (TBS): 50 mM Tris⋅Cl, pH 7.5/150 mM NaCl
  • Streptavidin‐POD conjugate (Roche Diagnostics)
  • Chemiluminescence Blotting Substrate kit (Roche Diagnostics) containing:
    • Luminescence substrate solution
    • Starting solution B
    • Blocking reagent
  • Washing buffer: TBS containing 0.1% (v/v) Tween 20
  • 10× maleic acid stock solution: 1.0 M maleic acid (Fluka)/1.5 M NaCl, pH 7.5
  • Tween 20 (e.g., Sigma)
  • Anti‐HA high‐affinity antibody (Roche Diagnostics)
  • Peroxidase‐conjugated rabbit anti‐rat immunoglobulins (Dako)
  • Beckman GS‐6R microcentrifuge with swing‐out rotor
  • Microcentrifuge
  • Pasteur pipets, sterile
  • SDS‐PAGE system (e.g., Mini Protean II, Bio‐Rad), including electroblotting devices (Bio‐Rad)
  • Plastic box
  • Whatman 3MM filter paper, 7 × 10 cm
  • Gel‐drying apparatus
  • X‐Omat AR film (Eastman Kodak)
  • Luminescence detector (e.g., Lumi‐Imager, Roche Diagnostics)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7) and SDS‐PAGE ( appendix 3F)

Support Protocol 1: Isolation and Analysis of RNA

  Materials
  • Patient blood collected in a plastic EDTA‐coated blood‐collection tube (Greiner) or cultured cells that are 80% to 90% confluent in 75‐cm2 tissue culture flask
  • Histopaque‐1077 (Sigma)
  • PBS, pH 7.8 (see recipe), sterile, 4°C
  • Cycloheximide (e.g., Sigma; optional)
  • RNAzolB (Campro Scientific) containing guanidinium thiocyanate and phenol
  • Chloroform
  • Isopropanol
  • 70% and 100% ethanol, 4°C
  • T 10E 0.1 buffer, pH 8.0: 10 mM Tris⋅Cl/0.1 mM EDTA, pH 8.0
  • 3 M sodium acetate ( appendix 2D; pH adjusted to 5.6 with acetic acid)
  • 1 M NaOH
  • SeaKem LE agarose (FMC Bioproducts)
  • 1× TBE buffer ( appendix 2D), sterile
  • 10 mg/ml ethidium bromide ( appendix 2D)
  • Formamide loading buffer ( appendix 2D)
  • RNA size and quality standards
  • 12‐ml white cap tubes
  • Beckman GS‐6R microcentrifuge and swing‐out rotor
  • Cell scraper, sterile
  • CEP‐swabs (Life Technologies)
  • Spin‐Ease extraction tubes (Life Technologies)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
CAUTION: Guanidinium thiocyanate is an irritant and phenol is toxic. Handle with care.NOTE: Keep all RNA samples ice cold.

Alternate Protocol 1: Radioactive Protein Truncation Test Using Coupled Transcription/Translation

  Materials
  • TnT T7‐Coupled Rabbit Reticulocyte Lysate System (Promega) containing:
    • TnT T7 RNA polymerase
    • TnT rabbit reticulocyte lysate
    • TnT reaction buffer
    • Amino acid mix minus leucine, minus methionine, and minus cysteine
    • Luciferase‐encoding control plasmid
    • Luciferase
    • Luciferase assay reagent
    • 5 mCi/ml [3H]leucine (160 Ci/mmol, Amersham)
  • 1.5 U/µl ribonuclease inhibitor (e.g., RNasin, Promega)
  • H 2O, sterile
  • 2× SDS sample buffer (see recipe)
  • 14C‐labeled (Amersham) or prestained (Bio‐Rad) protein molecular weight markers
  • Staining solution (see recipe)
  • Destaining solution (see recipe)
  • Dimethylsulfoxide (DMSO)
  • DMSO/PPO: 1 M 2,5‐diphenyloxazole in DMSO
  • Plastic box
  • Whatman 3MM filter paper
  • Gel‐drying apparatus
  • X‐Omat AR film (Eastman Kodak)
  • Additional reagents and equipment for reverse transcription, PCR, and SDS‐PAGE as in the nonradioactive protein truncation test (see protocol 1), agarose gel electrophoresis (unit 2.7), SDS‐PAGE ( appendix 3F), and autoradiography (e.g., CPMB APPENDIX )
NOTE: All chemicals, solutions, and enzymes should be stored according to the manufacturer's instructions.
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Figures

Videos

Literature Cited

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Key Reference
   Roest et al., 1993. See above.
  First publication of the principle of the protein truncation test.
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