Fish Protein Electrophoresis

Overview: 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 according to size using gel electophoresis. Because they also separate a mixture of known proteins on the same gel, they will be able to estimate the sizes of the unknown proteins. Because they also run samples of actin and myosin, students may be able to determine which of the bands from their fish protein samples are the muscle proteins actin and myosin. 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 Two of Fish Protein Electrophoresis

9:05 - Remove samples from refrigerator and thaw in hot water bath for five to ten minutes.

9:20 - Load your five samples plus one standard into six of the twelve lanes on the gel you are sharing. The wells can hold 20 uL if perfectly formed, but to avoid cross-contamination do not load more than 15 uL of sample per well.

Be aware that there can be up to two gels per apparatus. Also, keep in mind that your gel partner group cannot have the same molecular weight standards as your group does (Actin/Myosin and Kaleidoscope must be on each gel).

RECORD THE SAMPLE AND LANE NUMBER.

9:40 - Connect gel box to power supply and make sure it is set to 150 vdc constant voltage. Listen to voltage versus current safety issues.

10:00 - Check gel progress. Make sure buffer isn't leaking from top reservoir to bottom reservoir. This could cause overheating and even arcing.

10:50 - Check gel progress.

11:10 - Check gel and if dye front is far enough along, turn off power supply and disconnect leads.

11:20 - Prepare a staining box with distilled water and pry the gel plates apart. Prying the gel plates apart is the most challenging aspect of this Lab and not at all easy. Listen to instructor hints.
Add gel to water. Wash 3 x 5 minutes with distilled water. This helps to get rid of any detergent clinging to outside of gel. The detergent can compete with the protein for dye binding.

11:40 - Add 200 mL BioSafe Coomassie to the staining box with your gel.

5 pm Friday - after at least 12 hours, Kibak or Beech will pour off the Coomassie Staining Solution and rinse your gel once with distilled water. The gel will then incubate in distilled water with a folded kimwipe until next week. The negatively charged cellulose in kimwipes serves as an effective competitor for Coomassie dye binding and the gel should become very clear except where dye has bound to protein.

Write up... take careful notes on today's procedure and the lecture at the end of the sample preparation. You will need this information for your write-up due in two weeks after next week's analysis.