Fish Protein Electrophoresis

During the first period of this lab students remove muscle tissue from four species of fish and one invertebrate.  They extract some proteins from that tissue using detergent and prepare those extracts for electrophoresis.

During the second lab period, students separate the extracted proteins by size using gel electophoresis. The proteins in the gels are visualized by staining.

During the final lab period, students destain and photograph the gels.  They calculate the molecular weights of the dominant proteins and compare them to actin and myosin controls.  Finally they analyze the relationships between the species based on the pattern of proteins found in their muscle extracts.

Week One

For this lab, students will work in groups of two or three.   Make sure everything is labeled as indicated on the board.  LABELLING IS KEY TO SUCESS IN THIS LAB!  You will have to keep track of your work over a three week period.

• Read instructions completely.
• Obtain muscle samples from a variety of fish and one invertebrate.
• Extract and denature sample proteins.

1. Title, date, name your "protocol record sheet."
2. Use a black Sharpie to number each of the green1.5 mL flip-top eppendorf tubes you will be using.
3. Now write those same numbers down in a row in your lab notebook. Do you have a table of contents for your lab notebook yet? Might be a good idea to start one!
4. Next to those numbers write the name of the specimen that appears on the board.
5. Again use a Sharpie to number a corresponding set of clear eppendorf tubes. They will be used later.
6. Obtain a tube of Sample Preparation Buffer, (also known as Laemli Sample Buffer and labelled "LSB") from the front table and "spin it down." Spinning something down means to give it a quick pulse in the centrifuge to bring down and mix any condensation or drops that have collected on tube cap or wall during the freeze-thaw cycle.
7. Add 200 uL LSB carefully to each of the green flip-top eppendorf tubes you labeled and store the tubes nicely in your tube rack... "carefully" because we don't have extra and it can be difficult to pipet. Another reason to first "spin down."
8. Label your plastic disposable trays (weigh boats) with the names of the muscle specimen listed on the board.
9. Place a piece of muscle on the appropriate tray and cut away a very small (approximately 0.25 x 0.25 x 0.25 cm) cube of muscle tissue.  Avoid taking any fat, skin, or bones.
10. Transfer the tiny piece into the appropriately numbered green flip-top tube containing LSB and close the lid. Clean your tools between each sample type using kimwipes and a little soapy water.
11. When you have finished transferring all the pieces write down the time. Starting with the first tube, vortex each tube for 5 seconds at setting 6 and set it back in the rack.
12. All samples should sit in LSB for at least 5 minutes, and then be vortexed again.
13. After 5 minutes have elapsed for all samples, balance them in the microcentrifuge and "spin them down."
14. Set your p200 autopipet to 50 uL and carefully transfer 50 uL from each tube containing muscle to the appropriately labeled clear eppendorf tube.  Do not pull up any muscle into the pipet tip, and discard tips between samples. 
15. You will also receive one tube containing either the pre-stained molecular weight standards or the actin and myosin standards.  You will be assigned one or the other in class.  Number that screw cap tube #6 and record the appropriate information in your lab notebook.
16. Obtain a tube float rack and incubate your clear 1.5 mL eppendorf tubes (including one containing standards) at 90° C for five minutes.
17. Store the clear tubes at -20° C until next week.
18. Prepare to take notes from the instructor who will now provide an overview of the lab and background information.

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