1. Gel electrophoresis involves gelatinous support materials (agarose - for DNA or polyacrylamide - usually for proteins) and an electric current that drags molecules through the gel. Electrodes are arranged such that the "top" or beginning of the gel is where the negative electrode is placed and the positive electrode is placed at the bottom or end of the gel. DNA is negatively charged, so it is repelled away from the top and towards the bottom of the gel. Separation is on the basis of size. Large molecules travel slowest in the gel, whereas the small molecules travel fastest. DNA fragments appear as bands on a gel and bands can be excised separately from the other bands for further manipulation.2. SDS-PAGE is used to separate proteins on the basis of size. The key to this method is coating the proteins with SDS. This is a detergent that denatures the proteins and coats them evenly with a negative charge.
3. 2D gel electrophoresis involves two separations in two dimensions. The first involves separation on the basis of charge. The second involves placing the tube containing the separated charged molecules, adding SDS, and then running the contents on SDS-page. The resulting gel has molecules separated by size in the vertical direction and by charge in the horizontal direction.
4. Proteins can be broken into smaller pieces using enzymes like trypsin (cuts lysine or arginine) or chymotrypsin (cuts at tyrosine, tryptophan, or phenylalanine).
Highlights Enzymes I
1. Enzymes catalyze reactions up to a trillion times faster than the same reactions without any catalysts.
2. Enzymes work by reducing the activation energy required for a reaction to occur. The energy available to cells is called Free Energy (delta G). Because enzymes lower that "starting" energy requirement, they make the reaction easier to occur and thus speed them up.
3. Note also (important) that enzymes do NOT change the free energy difference between the beginning reactants and the end products. Thus, enzymes do not change the overall energy of a reaction - only the energy required for the transition state.
4. A "substrate" is a molecule bound by an enzyme which it catalyzes a reaction upon. Substrates bind specific binding sites on enzymes that resemble their structure. An "active site" of an enzyme is a site on an enzyme where the reaction it catalyzes occurs.
5. There are two models for enzyme action relevant for our consideration. The "lock and key" model proposes that enzymes act like a "lock" that only certain keys (substrates) fit. This model works well for describing the binding of substrates, but is not helpful (or accurate) for describing the mechanism of catalysis.
6. The "induced fit" model of enzyme action proposes that enzymes change in response to binding of substrate and that change is at least partly responsible for the catalysis that occurs on the substrate. Thus, the induced fit model says that enzymes change substrates (by catalysis) and that substrates change enzymes (enabling catalysis).
7. It is important to note that after catalysis occurs, the product is released and the enzyme is returned to its original state.