Detection and Manipulation of the Stress Response Protein Metallothionein

Sadikshya Bhandari1, Clare Melchiorre1, Kristen Dostie1, Debby Laukens2, Lindsey Devisscher2, Ariel Louwrier3, Amy Thees1, Michael A. Lynes1

1 Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, 2 Department of Internal Medicine, De Pintelaan, Gent, 3 StressMarq Biosciences, Victoria, British Columbia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.19
DOI:  10.1002/cptx.17
Online Posting Date:  February, 2017
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Abstract

Metallothioneins (MTs) are small molecular weight stress response proteins that play a central role as reservoir of essential divalent heavy metal cations such as zinc and copper, and also can diminish the effects of toxic heavy metals such as mercury and cadmium. Historically, MT has been considered to be an intracellular protein with roles to play in the management of heavy metals, as a regulator of cellular redox potential, and as a buffer of free radicals. Our recent studies have highlighted immunomodulatory role of MT in inflammatory diseases and also in the progression of metastatic cell movement. Hence, manipulation and detection of MT is essential for its possible use as a diagnostic and in therapeutic interventions of chronic inflammation. This review describes procedures used to detect MT using techniques such as western immunoblot, competition ELISA, flow cytometry and immunohistochemistry. Additionally, it also describes the use of a colorimetric cell proliferation assay (CellTiter 96 AQueous One Solution/MTS) to study the proliferative effect of MT. © 2017 by John Wiley & Sons, Inc.

Keywords: metallothionein; stress; inflammation; metal homeostasis

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Metallothionein Detection by Immunoblot
  • Basic Protocol 2: Metallothionein Detection by Competition ELISA
  • Basic Protocol 3: Detection of Metallothionein Cell Surface Binding and Intracellular Expression by Flow Cytometry
  • Basic Protocol 4: Metallothionein‐Mediated Splenocyte Proliferation Using CellTiter 96 AQueous One Solution/MTS Assay
  • Basic Protocol 5: Immunohistochemical Detection of Metallothionein on Paraffin‐Embedded Sections
  • Support Protocol 1: Sample Preparation for Metallothionein Detection
  • Support Protocol 2: Metallothionein Biotinylation
  • Support Protocol 3: Alexa Fluor 647 Conjugation to UC1MT
  • Support Protocol 4: Metallothionein Induction in Jurkat T Cells with Cadmium or Zinc Treatment
  • Alternate Protocol 1: Metallothionein Competition ELISA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Metallothionein Detection by Immunoblot

  Materials
  • Metallothionein (MT; Enzo Life Sciences, cat. no. ALX‐202‐072‐M001)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 6× loading buffer (see recipe)
  • 15% acrylamide gel
  • Running buffer (see recipe)
  • Full‐Range Rainbow Molecular Weight Marker (GE Healthcare, cat. no. RPN800E)
  • Pre‐transfer reducing buffer (see recipe)
  • Blocking Buffer (see recipe)
  • Tris‐buffered saline with Tween‐20 (TBS‐T)
  • UC1MT or E9 antibody (multiple suppliers)
  • Secondary antibody: Goat anti‐mouse Ig (H + L)‐AP (SouthernBiotech, cat. no. 1010‐04)
  • Substrate buffer (see recipe)
  • Distilled water
  • 1.5‐ml microcentrifuge tubes
  • Water bath
  • Mini‐PROTEAN Electrophoresis System (BioRad)
  • Benchtop centrifuge
  • Gel‐loading pipet tips
  • iBlot System (ThermoFisher Scientific)
  • 37°C incubator
  • iBlot Transfer Stack, PVDF mini (ThermoFisher Scientific, cat. no. IB401002)
  • Shallow dishes with lids
  • Shaker

Basic Protocol 2: Metallothionein Detection by Competition ELISA

  Materials
  • Coating buffer (see recipe)
  • UC1MT (Enzo Life Sciences, and other various sources)
  • Binding buffer (see recipe)
  • Metallothionein (MT; Enzo Life Sciences, cat. no. ALX‐202‐072‐M001)
  • Ice
  • MT‐biotin (see protocol 7 for MT biotinylation)
  • PBS‐T
  • Blocking buffer (see recipe)
  • Streptavidin‐alkaline phosphatase (R&D Systems, cat. no. AR001)
  • AP substrate buffer (see recipe)
  • p‐Nitrophenyl phosphatase tablets (pNPP; Sigma Aldrich, cat. no. N1891)
  • Immulon 2 HB 96‐well plate (Fisher Scientific, cat. no. 1424561)
  • 37°C incubator
  • Plate washer (BioTek, ELx405)
  • Vortex mixer
  • Microcentrifuge
  • Plate reader (Molecular Devices, Spectramax M2)

Basic Protocol 3: Detection of Metallothionein Cell Surface Binding and Intracellular Expression by Flow Cytometry

  Materials
  • Cells
  • FACS buffer (see recipe)
  • FIX and PERM Cell Permeabilization kit (ThermoFisher Scientific, cat. no. GAS004)
  • Normal goat serum (Sigma Aldrich, cat. no. G9023)
  • AlexaFluor647‐labeled UC1MT (see protocol 8)
  • AlexaFluor647‐MOPC21 (Biolegend, cat. no. 400130)
  • Ice
  • 12 × 75—mm glass or polystyrene tubes
  • Centrifuge
  • FACSCalibur flow cytometer

Basic Protocol 4: Metallothionein‐Mediated Splenocyte Proliferation Using CellTiter 96 AQueous One Solution/MTS Assay

  Materials
  • M199 complete medium (see recipe)
  • Ice
  • ACK lysis buffer (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Concanavalin A (Sigma, cat. no. C5275)
  • Lipopolysaccharide (LPS; Sigma, cat. no. L2630)
  • Metallothionein (MT; Enzo, cat. no. ALX‐202‐072‐M001)
  • Special gas (10% CO 2, 7% O 2, 83% N 2) (Airgas)
  • CellTiter 96 AQ ueous One Solution Cell Proliferation Assay (Promega, cat. no. G3582)
  • 1.5‐ml microcentrifuge tubes
  • Sterile blue pellet pestle; sized for microcentrifuge tube (multiple suppliers)
  • Cell strainer (Fisher Scientific)
  • Centrifuge
  • Shaker
  • Hemacytometer
  • 96‐well tissue culture treated plate
  • 37°C incubator with humidity
  • Plate reader (Molecular Devices, Spectramax M2)

Basic Protocol 5: Immunohistochemical Detection of Metallothionein on Paraffin‐Embedded Sections

  Materials
  • Xylene (Klinipath, cat. no. 4055)
  • 99% (v/v) ethanol (Klinipath, cat. no. 4098)
  • 96% (v/v) ethanol (Klinipath, cat. no. 4095)
  • Citrate buffer (see recipe)
  • 30% H 2O 2 (Sigma, cat. no. H1009)
  • Dulbecco's phosphate‐buffered saline (PBS), pH 7.4 (Invitrogen, cat. no. 14200)
  • Protein block (Dako, cat. no. X0909)
  • Fab fragment goat anti‐mouse IgG (Jackson ImmunoResearch, cat. no. 115‐007‐003)
  • Bovine serum albumin (BSA; Sigma, cat. no. A9647)
  • Mouse anti‐metallothionein (Invitrogen, clone E9)
  • Mouse IgG1 (Dako, cat. no. X0931)
  • LSAB2 visualization kit (Dako, cat. no. K0672) containing:
    • Biotinylated Universal link for 20′
    • Streptavidin‐peroxidase
  • Diaminobenzidine (DAB) + chromogen solution (Dako, cat. no. K3468)
  • Mayers Hematoxylin (Sigma, cat. no. MHS32)
  • Ultramount Aqueous Mounting medium (Dako, cat. no. S1964)
  • 95°C water bath
  

Support Protocol 1: Sample Preparation for Metallothionein Detection

  Materials
  • Μice
  • Ιce
  • Tissue lysis buffer (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe), ice‐cold
  • Triton X‐100 Lysis buffer with protease inhibitors
  • microBSA assay (Pierce)
  • 1.5‐ml microcentrifuge
  • Pellet pestle
  • Centrifuge
  • Microcentrifuge
  • Hemacytometer
  • Vortex mixer

Support Protocol 2: Metallothionein Biotinylation

  Materials
  • Metallothionein (MTl Enzo Life Sciences, cat. no. cat. no. ALX‐202‐072‐M001)
  • Potassium‐free phosphate‐buffered saline (PBS; see recipe)
  • Dimethyl sulfoxide (DMSO)
  • EZ‐Link NHS‐LC‐Biotin (ThermoFisher Scientific, cat. no. 21336)
  • Ice
  • HABA/Avidin Premix (ThermoFisher Scientific, cat. no. 28005)
  • ddH 2O
  • Biotinylated horseradish peroxidase (ThermoFisher Scientific, cat. no. 29139)
  • Zeba Spin Desalting Column, 2 ml (ThermoFisher Scientific, cat. no. 89889)
  • 15‐ml centrifuge tubes
  • Centrifuge
  • Pipets
  • 96‐well flat‐bottom microtiter plate
  • Shaker

Support Protocol 3: Alexa Fluor 647 Conjugation to UC1MT

  Materials
  • Purified UC1MT, known concentration
  • Phosphate‐buffered saline (PBS; see recipe)
  • Alexa Fluor 647 Protein Labeling kit (ThermoFisher Scientific ca. no. A20173) containing:
    • Alexa Fluor 647 reactive dye
    • Sodium bicarbonate
    • Purification resin
    • 10× elution buffer
    • Purification column
    • Column funnel
    • Foam column holder
    • Disposable pipet
    • Collection tubes
  • Vortex mixer, optional
  • Ring stand

Support Protocol 4: Metallothionein Induction in Jurkat T Cells with Cadmium or Zinc Treatment

  Materials
  • Complete RPMI (see recipe)
  • 10 mM stock solution of CdCl 2, filter‐sterilized
  • 50 mM stock solution of ZnCl 2, filter‐sterilized
  • Jurkat T cells (ATCC, cat. no. TIB‐152)
  • Hemacytometer
  • 1.5‐ml microcentrifuge tubes
  • 24‐well cell‐culture treated plate
  • 37°C, 5% CO 2 incubator

Alternate Protocol 1: Metallothionein Competition ELISA

  Materials
  • Metallothionein (MT; Enzo Life Sciences, cat. no. ALX‐202‐072‐M001)
  • Coating buffer (see recipe)
  • Binding buffer (see recipe)
  • Ice
  • UC1MT
  • PBS‐T
  • Blocking buffer (see recipe)
  • Goat anti‐mouse Ig (H + L)‐Alkaline Phosphatase (SouthernBiotech, cat. no. 1010‐04)
  • Phosphate‐buffered saline (PBS; see recipe)
  • AP substrate buffer (see recipe)
  • p‐Nitrophenyl phosphatase tablets (pNPP; Sigma Aldrich, cat. no. N1891)
  • Immulon 2 HB 96‐well plate (Fisher Scientific, cat. no. 1424561)
  • 37°C incubator
  • Vortex mixer
  • Plate washer (BioTek, ELx405) or micropipettor
  • Centrifuge
  • Plate reader (Molecular Devices, Spectramax M2)
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Figures

Videos

Literature Cited

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