1. Comparative Proteomics Kit I: Protein Profiler Module
Bio-Rad EDU
32 students
List Price: $ Refill: $ 94.00

2. 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,transfer pipets, gel-staining trays, test tube holders Teacher's Guide, Student Manual, and graphic Quick Guide 1

3. 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

4. Is There Something Fishy About Teaching Evolution?

5. 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

6. Workshop Timeline Introduction Sample Preparation
Load and electrophorese protein samples
Compare protein profiles
Construct cladograms

7. 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

8. Sample Preparation
Lab Period 1 Label one 1.5 ml fliptop tube for each of five fish samples. Also label one screwcap micro tube for each fish sample. Add 250 μl of Bio-Rad Laemmli sample buffer to each labeled fliptop microtube. 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!)

9. 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 screwcap tube. Do not transfer the fish! Heat the fish samples in screwcap microtubes for 5 minutes at 95°C. Freeze until lab period 2.

10. 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.

11. 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 μl actin and myosin standard (AM)

12. Electrophoresis
Lab Period 2 (con’t) Electrophorese for 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 Coomassie blue stain per gel and stain gel for 1 hour, with gentle shaking for best results.

13. 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.

14. 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.