Comparative Proteomics Kit I: Protein Profiler Module Bio-Rad 166-2700EDU 32 students List Price: $203.75 Refill: $ 94.00
Comparative Proteomics Kit I: Protein Profiler Module Laemmli sample buffer Kaleidoscope™ prestained standards Tris-glycine-SDS electrophoresis buffer Bio-Safe™ Coomassie stain for proteins Actin and myosin standard Dithiothreitol (DTT) Pipet tips for gel loading Test tubes,transferpipets, gel-staining trays, test tube holders Teacher's Guide, Student Manual, and graphic Quick Guide 1
Required Accessories Not Included in Kit Fish samples 5–8 types Adjustable micropipets, 2–20 µl Power supplies Water bath If using polyacrylamide gel electrophoresis:Vertical gel electrophoresis chambers Precast polyacrylamide gels
Explore Biochemical Evidence for Evolution • Analyze protein profiles from a variety of fish • Study protein structure/function • Use polyacrylamide electrophoresis to separate proteins by size • Construct cladograms using data from students’ gel analysis • Compare biochemical and phylogenetic relationships. • Sufficient materials for 8 student workstations • Can be completed in three 45 minute lab sessions
Workshop Timeline • Introduction • Sample Preparation • Load and electrophorese protein samples • Compare protein profiles • Construct cladograms
Can biomolecular evidence be used to determine evolutionary relationships? • Traits are the result of Structure and Function • Proteins determine structure and function • DNA codes for proteins that confer traits • DNA -> RNA -> Protein -> Trait • Changes in DNA lead to proteins with: • Different functions • Novel traits • Positive, negative, or no effects • Genetic diversity provides pool for natural selection = evolution
Sample Preparation Lab Period 1 Label one 1.5 ml fliptoptube for each of five fish samples. Also label one screwcapmicro tube for each fish sample. Add 250 μl of Bio-Rad Laemmli sample buffer to each labeled fliptopmicrotube. Cut a piece of each fish muscle about 0.25 x 0.25 x 0.25 cm3 and transfer each piece into a labeled fliptop tube. (Close the lid!)
Sample Preparation Lab Period 1 (con’t) Flick the microtubes 15 times to agitate the tissue in the sample buffer. Incubate for 5 minutes at room temperature. Carefully transfer the buffer by pouring from each fliptop tube into a labeled screwcaptube. Do not transfer the fish! Heat the fish samples in screwcapmicrotubes for 5 minutes at 95°C. Freeze until lab period 2.
Electrophoresis Lab Period 2 Heat extracted fish samples and actin and myosin standard to 95°C for 2–5 min. This dissolves any detergent in the extraction (Laemmli) buffer that may have precipitated upon freezing.
Electrophoresis Lab Period 2 (con’t) Load your gel: • 5 μl Precision Plus Protein Kaleidoscope prestained standards (Stds) • 10 μl fish sample 1 • 10 μl fish sample 2 • 10 μl fish sample 3 • 10 μlactin and myosin standard (AM)
Electrophoresis Lab Period 2 (con’t) Electrophoresefor 30 minutes at 200 V in 1x TGS electrophoresis buffer. After electrophoresis, remove gel from cassette and transfer gel to a container with 25 ml Bio-Safe Coomassieblue stain per gel and stain gel for 1 hour, with gentle shaking for best results.
Electrophoresis Lab Period 3 Discard stain and destain gels in a large volume of water for at least 30 minutes to overnight, changing the water at least once. Blue-stained bands will be visible on a clear gel after destaining. Dry gels using GelAir cellophane.
Analysis Lab Period 3 (con’t) Correlate bands of fish samples with AM and Kaleidoscope standards. Check online protein database for correlation of sample proteins with those of the species in the databases. Guided by similarity of protein content, draw cladogram relating fish species.