The purpose of this experiment is to determine the molecular weights of the major plasma membrane proteins of mammalian red blood cells using SDS-PAGE.
1. Sample Preparation
a. Label a microcentrifuge tube with your initials.
b. Using an automatic pipettor, pipet a volume of your plasma membrane sample equivalent to 100 µg of protein into the labeled microcentrifuge tube (normally this is approximately 50 µl).
c. Using an automatic pipettor, add 100 µl electrophoresis sample buffer to the sample. Cap and vortex the tube for 15 seconds.
d. Place the tube in boiling water for 5 minutes. Retrieve the tubes and allow to cool.
2. Load Your Sample Onto the Gel
a. Load 10 µl of your sample into each of two sample wells at the top of your the gel as demonstrated in class.
b. Your instructor will load the standards. The standards are known proteins of known molecular weights. To find out more about the importance of standards, turn to the next page.
3. Run the Gel
a. Once all samples have been loaded, place the lid on the electrophoresis chamber. Attach the leads to the power supply. When assembled properly, the anode will be at the top of the gel, the cathode will be at the bottom. Apply power. The recommended power for these gels is 200 volts.
b. Your team needs to monitor the migration of your samples through the gel. When the solvent front migrates to within 0.5 cm of the bottom of the gel, the run is over. Turn off the power supply. The usual run time is approximately 35 - 40 minutes.
4. Stain the Gel
Remove the gel from the electrophoresis apparatus. Place the gel in a staining tray and proceed to stain the gel according to the staining protocol outlined in class. Staining takes some time, and might require some out-of-class lab work.
5. Photograph the Gel