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now: Sat Jun 6 12:54:56 2020 ... mod: Mon Oct 12 05:09:13 2015
Chris, I believe there is aclautly a small amount of weakness in the toxicological defense per Paracelsus. In his time he was speaking of the effects of poisoning as carcinogenic effects were pretty much unknown as a class.Today scientists are divided between the threshold and non-threshold theory camps for carcinogenesis, with the non-thresholders claiming that even a single exposure to the smallest possible immeasurable unit of a substance or condition can cause cancer.There are two problems with this however:1) Many scientists disagree with the theory, claiming that something akin to chaos theory or prinicples of uncertainty enter the equation significantly when the exposures become too small. These scientists claim that a butterfly flapping its wings in Kansas can NOT ever "cause" a typhoon in Vietnam.2) Even if we accept the no-threshold theory the Antismokers' characterization of secondary smoke is misleading. To use the word toxic or dangerous to describe the level of exposure to secondary smoke that would normally be encountered today in any decently ventilated establishment would be like using those words to describe reaching out the door to grab the morning paper without UV safety gloves on or dining in a restaurant where someone was drinking a beverage containing the highly volatile Class A Human Carcinogen ethyl alcohol.Use of "no safe level" to describe those exposures would be highly misleading and destructive.... just as its use to describe secondhand smoke is.Michael J. McFadden??
Recipes for the Bio-Rad Mini Protean III SDS-PAGE system
See also the long MiniProteanThreeInstructions without recipes.
The percentage of the resolving gel determines the molecular weights of proteins which can be resolved. 12% is a good generic gel to use and will resolve proteins from 15 kDa to 200 kDa with emphasis on resolving proteins from 15 kDa to 100 kDa. Lower percentage resolving gels will do a better job of resolving larger proteins, but will allow smaller molecular weight (MW) species to migrate off the gel. Higher percentage gels will similarly give extra resolution to lower MW species at a sacrifice of higher MW resolution.
|resolving gel || 6% || 8% || 10% || 12% |
|40% acrylamide (29:1 crosslink) || 600 μl || 800 μl || 1.0 ml || 1.2 ml|
|4x resolving buffer pH 8.8 || 1 ml || 1 ml || 1 ml || 1 ml|
|H2O || 2.4 ml || 2.2 ml || 2.0 ml || 1.8 ml|
|10% APS || 20 μl || 20 μl || 20 μl || 20 μl|
|TEMED || 2 μl || 2 μl || 2 μl || 2 μl|
|Total volume || 4.0 ml || 4.0 ml || 4.0 ml || 4.0 ml|
- actually use 3.5 ml per gel
- carefully layer water on top of the liquid gel after it has been poured and before the acrylamide has polymerized to ensure an evenly flat top to the stacking gel
- polymerize for 45 minutes to 1 hour
- do not use less time even if the gel appears to have completed polymerization
|stacking gel || one gel || two gels|
|40% acrylamide (29:1 crosslink) || 117 μl || 234 μl|
|4x stacking buffer pH 6.8 || 300 μl || 600 μl|
|H2O || 783 μl || 1566 μl|
|10% APS || 12 μl || 24 μl|
|TEMED || 2 μl || 4 μl|
|Total volume || 1200 μl || 2400 μl|
- actually use about 1 ml of mix per gel
- be very careful to eliminate all air bubbles since oxygen inhibits acrylamide polymerization
The Mini Protean III needs about 350 ml of running buffer:
- be sure to rinse out the wells with running buffer after you assemble the gel into the running apparatus
- boil samples in SDS loading buffer for two minutes and give a quick microcentrifuge spin-down immediately prior to loading
- run the gel at 200 V constant voltage until the tracking dye has just finished migrating out of the bottom of the gel