There’s nothing FISHY about evolution.  Is there?

Evolution is the change in the gene pool over time.
Since  DNA > RNA > PROTEIN >TRAIT
then a change in the gene pool = changes in DNA = changes in proteins.

Investigate evolution scientifically.
Bio-Rad’s new Biotechnology Explorer Kit allows you to explore evolution critically using protein electrophoresis and hands-on, inquiry-based learning. Studying DNA is sexy and fashionable, but the ultimate function of DNA is to make proteins. Explore the complete molecular framework of Biology: from DNA to RNA to PROTEIN to TRAIT. 

Compare protein profiles in related or divergent fish. Can biomolecular evidence be used to determine similarities and differences among species? You and your students will be amazed at what you discover!

Protocol for fish muscle protein extraction and sample preparation
For each fish to be analyzed: (e.g., catfish, trout, salmon, shark, sturgeon…)

1.      Label a 1.5 ml flip-top microtube for each fish sample being prepared.
2.      Add 500 microliters of Bio-Rad Laemmli Sample Buffer to each labeled tube and close the lid. Use Laemmli Sample          Buffer straight from the bottle. (Addition of beta-mercaptoethanol (BME) is neither required nor recommended for this       activity).
3.      Cut a piece of fish muscle (avoid skin, fat and bones) that weighs 0.15 g or has dimensions of 0.5 x 0.5 x 1.0 cm (about       the size of a Tic-Tac™) and transfer into the appropriately labeled microtube. 

4.      Flick the microtube 15 times with your finger to agitate the tissue in the buffer

5.      Incubate 5 minutes at room temperature to solubilize proteins.

6.      Transfer the buffer containing the extracted proteins to a labeled 1.5 ml screw-cap tube by pouring the buffer from the flip-top tube into the screw-cap tube.

Note: It’s not necessary to transfer the entire volume to the screw cap tube.  Only a small volume is actually needed for gel loading. 

7.      Heat the sample in the screw-cap tube for 5 minutes at 95 ^oC to denature proteins before electrophoresis.

8.      Samples can be stored at room temperature if gels are loaded within several hours or stored at –20^oC for several weeks.

 SDS-PAGE gel loading and running:

 1.      Load gel, centering samples.  For 5 fish samples on a 10 well gel, you may choose to follow this guide:

 2.  To load each sample, use a thin, protein gel loading micropipette tip to withdraw 10 microliters of each sample and gently transfer it into the indicated gel well.

3.  When all samples have been loaded, run the gels for 30 minutes at 200V in 1X Tris-glycine-SDS running buffer.  (Dilute 1 part 10X TGS buffer in 9 parts distilled water to make a 1X working solution).

Gel staining and drying:
1.  When gels are finished running, carefully remove each gel from its cassette and transfer to a container with a large         volume of water. Gently shake the container for 5 minutes.  Carefully discard water, replace with fresh water, and         repeat this wash two more times.
   Note:  If time is limited, gels can be transferred directly into BioSafe Coomassie Stain, without the water rinses.

2.  After the last wash, remove as much water as possible from the container and stain the gels with 50 ml BioSafe Blue Coomassie Stain for 1-8 hours, with shaking for best results.

 3.  Destain the gels at least 30 minutes (overnight is ideal) with several changes of a large volume of water.  Blue bands        should be clearly visible on a clear background. Gels can be stored in water for up to one week before drying. 

 4.  Dry gels using GelAir cellophane sandwich and Tupperware™ container (or GelAir gel drying frames).
      Note: For best results, trim off the thick bottom edge of the gel before drying.

Wet two pieces of cellophane in a large volume of water.  Place one sheet of cellophane over a Tupperware™ or staining container.  Pull cellophane taut so that it makes a flat plane over the top of the container.  Use a rubber band to secure the cellophane in place. Place gel onto the cellophane.  Be sure to remove any air bubbles that may lie under or around the gel.  Flooding the surface of the cellophane around the gel with water will aid in the removal of air bubbles.  Place the second sheet of wetted cellophane over the gel, being careful not to trap any air bubbles.  Secure the second sheet of cellophane to the box with a second rubber band.  Allow gel to dry several days in a well-ventilated area.

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