Introduction
Review the theory behind the SDS-PAGE technique (Pg 98). For today's lab, you will be pouring your own gel. The plates of the mini gel are assembled with 1.5 mm spacers and tightened onto the pouring base. When properly assembled, the apparatus will not leak. When preparing the ge solutions, it is imperative that the ammonium persulfate (APS) is added last because it will initiate the polymerization reaction. The resolving gel is poured first, since it is the lower portion of the SDS- PAGE gel. An alcohol solution is layered on top of the liquid resolving gel to exclude oxygen and ensure a flat upper edge to the resolving gel upon polymerization. Once the resolving gel is polymerized, a distinct line can usually be seen at the alcohol/gel interface. The alcohol layer is then poured off of the resolving gel. The stacking gel is poured on top of the resolving gel and a comb is inserted to form the wells for sample loading. It is necessary to fill the space completely with stacking gel solution in order to yield wells with maximum depth for sample loading. Once the stacking gel is polymerized, the comb is removed carefully to avoid breaking the wells. Note that due to the low percentage of acrylamide in the stacking gel, the thin walls of the wells often shift considerably and may have to be manually adjusted. The gel apparatus is then placed into the electrophoresis tank with running buffer.
Comparison of samples from each step of the ADH isolation procedure using SDS-PAGE provides another way to tracking the purity of the enzyme during purification. The ADH enzymatic assays and specific activity calculations have provided some evidence of the level of purity of the final samples, but a stained SDS-PAGE gel of all samples allows for direct visualization of the proteins within the samples
Pre-lab
1. Thoroughly read the experimental procedures for the lab.
2. Provide a 2-3 sentence description of the purpose of the experiment.
3. Provide a 2-3 sentence description of the experimental procedure
4. Calculate the percentage of acrylamide in the resolving gel.
5. Describe your expected results for the protein patterns for this SDS-PAGE gel
6. What activity readings will be measured today?
SDS-PAGE Protocol.
WARNING: Aerylamide in the monomerie form is a neurotoxin. Always wear gloves when using this reagent and never mouth pipette solutions of the compound. All skin contact with the monomer should be washed off as soon as possible. After the acrylamide has polymerized it does not present a great hazard. If you have any coneerns or questions see your TA. Take care to properly dispose of anything that has come into contact with the acrylamide solutions (i.e. sleeve all pipettes) to minimize contamination of your work area with acrylamide. Be sure to wipe down your pipettors.

Introduction Review the theory behind the SDS-PAGE technique (Pg 9...