SDS-PAGE of Fish Muscle

Cut a 2mm cube of fish muscle and transfer it into a microcentrifuge tube containing 250 microliters of Laemmli sample buffer. Flick the tube to agitate the fish and incubate at room temperature for 5 minutes.

Transfer the buffer containing proteins into a screw cap tube, avoiding transfer of fish tissue. Firmly cap the tube and heat it for 5 minutes at 95°C to denature the proteins.

Remove the gel from packaging, peel off the bottom tape, and remove the comb. Set up the vertical electrophoresis system by placing gels into the electrode assembly with short plates facing inward, securing with green clamps, and lowering the assembly into the tank.

Fill the electrode assembly chamber with 1x TGS electrophoresis buffer to cover the gel short plates, then fill the outer tank to the four-gel line. Heat the fish sample and standards to 95°C, then load 5 microliters of protein standards in lane 1 and 10 microliters of fish sample in remaining lanes.

Place the lid on the chamber, connect electrical leads (red to red, black to black), and set voltage at 200 volts for 30 minutes. Monitor for bubble release, tracking dye progression, and protein separation as electrophoresis proceeds.

Turn off power and remove the chamber lid and electrode assembly. Release the green clamps, remove the gel cassette, and use the opening key to carefully separate the cassette and extract the gel.

Transfer the gel to a tray and rinse three times with tap water for 5 minutes each. Add 50 milliliters of Bio-Safe Coomassie stain and incubate for at least 1 hour, then rinse twice and destain overnight in a large volume of water with at least one water change.