Comparative Proteomics Kit II: Western Blot Module

Print
Comparative Proteomics Kit II: Western Blot Module

Overview

Lab Preparation Checklist
Kit contains sufficient materials for 8 student workstations. (2–4 students per workstation)
Primary antibody (anti-myosin light chain mouse monoclonal), lyophilized 1
Secondary antibody (goat anti-mouse polyclonal antibody conjugated to
horseradish peroxidase (HRP), lyophilized
1
HRP color detection reagent A
1
HRP color detection reagent B 1
10x Tris-glycine, 1 L 1
Non-fat dry milk blocker 1
10x Phosphate buffered saline (PBS), 100 ml 2
10% Tween-20, 5 ml 1
Nitrocellulose, 0.45 μm 8 sheets
Blotting paper 16 sheets
Reagent tubes 25
Curriculum, including Teacher's Guide, Student Manual, and graphic Quick Guide 1
Required Accessories Not Included in Kit:  
Comparative Proteomics Kit I: Protein Profiler Module 1
Ready Gel precast polyacrylamide gels, 15%, 10-well each 8
Vertical gel electrophoresis chambers 4
Adjustable micropipet, 2–20 µl 4–8
Power supplies 2–4
Water bath 1
Rocking platform 1
Reagent alcohol or ethanol, 0.8–2 L  
Fish samples 5–8 types
Recommended (Optional) Accessories:  
Electroblotter 4
Western Blot Flowchart
Western Blot Flowchart
Key Kit Features
  • Explore immunodetection
  • Explain HIV detection
  • Apply immunology
  • Antibodies as tools
  • Complete in four 45 minute lab sessions
Curriculum Fit
Comparative Proteomics Kit II Curriculum Fit
Western Blotting and Antibodies

Western blotting employs antibodies to pinpoint specific proteins of interest in complex protein mixtures such as cell extracts. Because of its accuracy, western blotting is used as the confirmatory diagnostic test for HIV and mad cow disease (bovine spongiform encephalopathy). Western blotting is used extensively in research to determine the presence of specific proteins, to quantify their expression levels, and to determine whether they have undergone genetic or posttranslational modifications. This surefire method categorically identifies proteins of interest based on two distinguishing features: molecular mass and antibody binding specificity.

When foreign invaders are detected, animal immune systems naturally generate antibodies and tag them for destruction. The ability of antibodies to act like magic bullets and target viral, bacterial, and allergenic antigens in the body also makes them ideal tools in the hunt for specific molecules in bioscience research and diagnostic tests.

Western blot of protein profiler gel reveals precise location of myosin light chain proteins.

PowerPoint Presentation for Workshops and for Teaching

Comparative Proteomics Kit II: Western Blot Module

This PowerPoint presentation is intended for educational use only. The presentation provides educators with background on the Comparative Proteomics Kit II: Western Blot Module and includes instructions for using the kit.

Comparative Proteomics Kit II: Western Blot Module PowerPoint presentation
Comparative Proteomics Kit II: Western Blot Module

The Comparative Proteomics Kit II: Western Blot Module is the second part of a two-part laboratory. In the first part of this laboratory (Comparative Proteomics Kit I: Protein Profiler Module), students use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to generate protein profiles and to visualize the unique array of proteins comprising muscle tissues from different fish. In this second part of the laboratory, students apply western blotting techniques to their polyacrylamide gel results to specifically identify myosin light chain from the hundreds of proteins that comprise the muscle cell extracts of closely and distantly related species of fish.

Features and Benefits

  • Applies immunology
  • Teaches students to use antibodies as detection tools
  • Serves as a laboratory extension for the Comparative Proteomics I Kit: Protein Profiler Module

How It Works

The protocol involves transferring the protein bands from polyacrylamide gels to a membrane and then using an anti-myosin light chain antibody to precisely identify which protein in each species' profile is myosin light chain. Using Internet-based bioinformatics databases, students can compare their experimentally determined results to actual protein sequence data derived from DNA and RNA sequences and consider whether variations in myosin among species are due to "genetic" or "epigenetic" factors.

More Information

  • Four-session laboratory activity, 45 min per session
  • Provides sufficient materials for eight student workstations, up to four students per workstation
Kit contains sufficient materials for 8 student workstations (2–4 students per workstation)  
Primary antibody (anti-myosin light chain mouse monoclonal), lyophilized 1
Secondary antibody (goat anti-mouse polyclonal antibody conjugated to horseradish peroxidase (HRP), lyophilized 1
HRP color detection reagent A 1
HRP color detection reagent B 1
10x Tris-glycine, 1 L 1
Non-fat dry milk blocker 1
10x phosphate buffered saline (PBS), 100 ml 2
10% Tween 20, 5 ml 1
Nitrocellulose sheets, 0.45 µm 8
Blotting paper sheets 16
Reagent tubes 25
Curriculum, including Teacher's Guide, Student Manual, and graphic Quick Guide 1
Comparative Proteomics Kit II: Western Blot Module

166-2800EDU
Western blot classroom study kit, includes antibodies, color detection reagents, 10x TG buffer, 10x PBS, 10% Tween, nitrocellulose, curriculum, and more, for 32 students; education use only

List Price:   Inquire
Quantity:   Add to Quote
Add to Hot List  
Comparative Proteomics Kits I and II: Protein Profiler Module and Western Blot Module

166-2850EDU
Comprehensive protein classroom study kit, includes Protein Profiler Module (#166-2700EDU) and Western Blot Module (#166-2800EDU), for 32 students; education use only

List Price:   Inquire
Quantity:   Add to Quote
Add to Hot List  
Rapid Blotting and V3 Western Workflow™ Starter Kit

166-2875EDU
Comprehensive protein classroom study kit, includes Protein Profiler Module (#166-2700EDU), Western Blot Module (#166-2800EDU), Trans-Blot® Turbo™ mini nitrocellulose transfer packs (10), 4–20% Mini-PROTEAN® TGX Stain-Free™ gels (10), application note, for 32 students; education use only

List Price:   Inquire
Quantity:   Add to Quote
Add to Hot List  
Number Description Options
M1662876EDU Biotechnology Explorer Rapid Blotting + V3 Western Workflow (Stain-Free Rapid Blotting) Application Note, Rev A Click to download
10004531 Instruction Manual, Comparative Proteomics Kit II: Western Blot Module, Biotechnology Explorer, Rev D Please login to download this document [ Add to Cart (Free) ]
5478 Instruction Manual, Comparative Proteomics Kit II: Western Blot Module Quick Guide, Biotechnology Explorer, Rev A Click to download