BISC220/S10: Mod 1 Lab 4

Polyacrylamide Gel Electrophoresis

Another way to determine the effectiveness of our purification technique is to perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on the CE and PF. SDS, an anionic detergent, is used to both denature the proteins and to give them an overall negative charge. The electrophoresis loading and running buffers also contain SDS. This negative charge allows the proteins to run toward the anode (positive electrode). The larger molecules are retarded by the acrylamide, which acts like a sieve, so they move more slowly through the gel than the smaller molecules. By running a sample of known molecular weights in a lane adjacent to the extracts, it is possible, by comparison, to determine the molecular weights of the bands of unknown proteins in each of the extracts. In addition to SDS, a denaturing and loading solution called Laemmli buffer contains:

1. 10% glycerol, which makes the solution dense enough to fall through the running buffer into the wells of the gel, so the sample doesn't float away;
2. 5% β−mercaptoethanol to break sulfide bonds and to keep disulfide bonds from reforming between the denatured proteins
3. 2% SDS Sodium dodecyl sulfate,
4. a trace amount of tracking dye, bromphenol blue, which will run ahead of the proteins, forming a visible front as the samples run during electrophoresis.
5. in the solvent 0.0625 M Tris buffer pH 6.7

The gels are placed in the tank, which is half filled with cold running buffer. The top chamber is filled and checked for leaks, which can also cause distortion of the current. The current runs from the top chamber through the gel to the lower chamber, which is connected to the anode (red electrode). The negatively charged proteins will migrate toward the anode according to their molecular weights with the smaller proteins migrating farther down the gel than the larger proteins.

Protocol

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