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 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 Three

2:00/9:00 - Listen to the mini-lecture on the concept of standards. Also, record the methods that were used to destain your gels in your absence. 

Obtain a sheet of plastic wrap at least 22 cm x 22 cm.

Remove your gel from the distilled water and place it on the sheet of plastic wrap without causing wrinkles. Throw away the kimwipe.

Prepare a table listing the distances traveled in millimeters of each band in each lane, including the standards.

Using semi-log graph paper, plot the migration of each of the prestained standards in millimeters against the molecular weight indicated in the handout (the apparent MW of the prestained standards varies from Lot to Lot due to inconsistent amounts of dye being bound to the individual proteins).

Begin the work of estimating the molecular weights of myosin, actin, and the bands of the fish proteins.

Prepare a table listing the molecular weights of bands found in each of the lanes for the samples.

Review your notes on character tables and relating species through networks.  Begin to think about how to represent these relationships in your write-up.

Be sure to pick up all the handouts.  You will need them for the materials and methods sections of your write-up.

Photograph the gel on the lightbox using the digital camera... magnify the gel until the distance between the bottom of a well and the dye front is just less than the image size.

Save your photograph the the class folder on the wet lab computer.

For the write-up...  

Prepare a graph on semi-log paper illustrating the migration distance and mass of your Kalaidescope standard proteins. Kalaidescope standards are modified proteins, so the molecular mass may be different than native proteins.

Use your graph to prepare a table of results recording the molecular weight of each band you can detect in your sample lanes, including the Actin and Myosin standard lane.

Identify the molecular weights of Actin and Myosin from each of the species and describe the differences in the distribution of protein bands extracted by this method from muscle tissue of each species. 

Prepare a table of "traits" for each species and develop a phylogenetic tree or network relating the five species. In other words, which two species are most closely related according to your data, which is the next most closely related, and so on...

For more details on how to think about phylogenetic trees or networks see your textbook, BIOSKILLS 2, page B-3 and pages 543-548 (Biological Science by Scott Freeman, 3rd Edition), or refer to your notes from Bio 240.