Comparative Proteomics Kit I: Protein Profiler Module

Bio-Rad 166-2700EDU32 students

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Comparative Proteomics Kit I: Protein Profiler Module

Laemmli sample bufferKaleidoscope™ prestained standardsTris-glycine-SDS electrophoresis bufferBio-Safe™ Coomassie stain for proteinsActin and myosin standardDithiothreitol (DTT)Pipet tips for gel loadingTest tubes,transfer pipets, gel-staining trays, test tube

holdersTeacher's Guide, Student Manual, and graphic Quick Guide 1

Required Accessories Not Included in Kit

Fish samples 5–8 typesAdjustable micropipets, 2–20 µlPower suppliesWater bathIf using polyacrylamide gel electrophoresis:

Vertical gel electrophoresis chambersPrecast polyacrylamide gels

Is There Something Fishy About Teaching Evolution?

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 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!)

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.

Electrophoresis

Lab Period 2Heat 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 μl actin and myosin standard (AM)

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.

Electrophoresis

Lab Period 3Discard 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.