Western blotting employs antibodies to identify proteins of interest in complex protein mixtures such as cell extracts and is a cornerstone of biomedical research.
- Explore a technique often used in medical diagnosis
- This exercise can not be performed without first completing the Bio-Rad® comparative proteomics kit I
- Time Required: Four, 45 min. lab sessions
This western blotting activity takes the protein profiler kit to the next level. Students generate protein profiles from the muscle tissue of closely and distantly related fish and then use the power of western blotting and immunodetection to specifically identify myosin from the hundreds of other muscle proteins in each fish's profiles. From their protein profiler results, students make educated guesses as to the identities of the proteins in their gels. Via western blotting, antibodies confirm the presence and location of one specific protein, myosin light chain, in each species profile. Measurable differences in the molecular weights of the myosin light chain proteins from different species lead students to hypothesize about how these variations relate to evolutionary relationships of the species.
Primary antibody (anti-myosin light chain mouse monoclonal), lyophilized
Secondary antibody (goat anti-mouse polyclonal antibody conjugated to horseradish peroxidase, or HRP), lyophilized
HRP color detection reagent A
HRP color detection reagent B
10x Tris/Glycine, 1 l
Nonfat dry milk blocker
10x Phosphate buffered saline (PBS), 100 ml
10% Tween 20, 5 ml
Nitrocellulose sheets, 0.45 μm
Blotting paper sheets
Curriculum including teacher's guide, student manual, and graphic quick guide
Delivery information: Includes reagents needed for western blotting and transfer. Required but not provided: Comparative proteomics kit, vertical gel electrophoresis equipment and transfor modules, 2 - 20 µl pipette, water bath, rocker, reagent alcohol, fish samples (5 - 8 types).
Kit will be shipped immediately. Must be stored appropriately upon receipt.