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Bio-Rad Comparative Proteomics Kits: Western Blot Module

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PLEASE NOTE: this kit can only be used with polyacrylamide vertical gel electrophoresis equipment. Please contact your territory manager for further information and a full list of the required components.

Animal immune systems respond to invasion by generating antibodies, which tag the foreign material 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 for hunting antigens in bioscience research and diagnostic tests.

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, or 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 post-translational modifications. This sure-fire method categorically identifies proteins of interest based on two distinguishing features: molecular mass and antibody binding specificity.

The western blot kit activity allows your students to take Bio-Rad s protein profiler kit to the next level. Students use western blotting to specifically identify myosin light chain proteins from the hundreds of other proteins that comprise the muscle cell extracts of closely and distantly related species of fish.

In the first part of this laboratory (Protein Profiler Module, EPE 120 080), students generate protein profiles and visualise the unique arrays of proteins comprising the muscle tissues from each of their samples. From protein gel results, students make educated guesses as to the identities of the proteins. Based on relative molecular masses alone, however, these inferences remain guesses. Via western blotting, the protein bands in the students polyacrylamide gels are transferred horizontally to a membrane, and an anti-myosin antibody is employed to precisely identify which protein in each species profile is myosin light chain.

Myosin is a major muscle protein essential for locomotion and survival in all animals. As such, the essential structure and function of myosin has remained relatively stable or 'conserved' in all animals over evolutionary time. However, differences in the molecular weights of the myosin light chains of different species are detectable via western blotting, leading students to hypothesise about how these variations relate to evolutionary relationships. Using Internet-based bioinformatics databases, students can then compare their experimentally determined results to actual protein sequence data derived from DNA and RNA sequences and consider whether variations in myosins between species are due to genetic or epigenetic factors.

Key Kit Features:
● Explore immunodetection
● Explain HIV detection
● Apply immunology
● Employ antibodies as tools
● Complete in four 45-minute lab sessions

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Associated Products

Bio-Rad Comparative Proteomics Kits: Protein Profiler Module

Catalogue Code: EPE120080
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Catalogue Price: £144.69

Bio-Rad Comparative Proteomics Kits: Combined Kit

Catalogue Code: EPE120100
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Catalogue Price: £399.81
DescriptionWestern Blot Module
● Primary antibody (anti-myosin light chain mouse monoclonal), lyophilized, 1 vial
● Secondary antibody (goat anti-mouse polyclonal antibody conjugated to horseradish peroxidase
● (HRP)), lyophilized, 1 vial
● HRP colour detection reagent A, 1 bottle
● HRP colour detection reagent B, 1 vial
● Buffer, 10x Tris/glycine, 1 bottle
● Nonfat dry milk blocker, 1 pack
● 10x Phosphate buffered saline (PBS), 2 bottles
● 10% Tween 20, 1 bottle
● Nitrocellulose membranes, 0.45 µm, 8 sheets
● Blotting paper, 16 sheets
● Reagent tubes, 1 pack
● Curriculum/Instruction manual