Glycerol buffer, solution preparation

Repeat step 6-20 with the exception that the 10 mg samples should be dissolved in 3 ml 0. IM phosphate or borate buffer adjusted to pH 9.0. 6-52. For each of the protein solutions prepared in step 6-51, prepare an electrophoretic sample solution composed of 0.01 ml 50% aqueous glycerol, 0.020 ml of the solution prepared in step 6-19, and 0.005 ml of the protein solution prepared in step 6-51. 

The most commonly used combination of chemicals to produce a polyacrylamide gel is acrylamide, bis acrylamide, buffer, ammonium persulfate, and tetramethylenediarnine (TEMED). TEMED and ammonium persulfate are catalysts to the polymerization reaction. The TEMED causes the persulfate to produce free radicals, causing polymerization. Because this is a free-radical driven reaction, the mixture of reagents must be degassed before it is used. The mixture polymerizes quickly after TEMED addition, so it should be poured into the gel-casting apparatus as quickly as possible. Once the gel is poured into a prepared form, a comb can be appHed to the top portion of the gel before polymerization occurs. This comb sets small indentations permanently into the top portion of the gel which can be used to load samples. If the comb is used, samples are then typically mixed with a heavier solution, such as glycerol, before the sample is appHed to the gel, to prevent the sample from dispersing into the reservoir buffer. 

The SI70 supernatant (220 ml) was made to 40 % saturation with solid ammonium sulfate, stirred for 20 min, and then the precipitate was collected by centrifugation at 15,000 g for 15 min. The precipitate was suspended in small volume of buffer B-50 at pH 7.6 containing 20 mM HEPES/KOH, 0.1 mM EDTA, 1 mM dithiothreitol, 10 % (v/v) glycerol, and 50 mM potassium acetate. The 60 % saturated ammonium sulfate solution was prepared similarly. Protein concentrations for 0 - 40 % and 40 - 60 % ammonium sulfate fractions were 4.2 mg/ml and 4.7 mg/ml, respectively. 

Prepare the sample in buffer E with a protein concentration not more than 20 mg/ml. Heat the solution to 95 °C for 2-3 min and supplement with some crystals of sucrose or a droplet of glycerol or 1/10 volume of bromophenol blue in 50% sucrose solution. 

For glycerol stock, 50 pL of the culture is added to 25 pL of Cell Stock Buffer in a 96-well plate. The remaining suspensions are applied to plasmid preparation using the Wizard SV 96 Plasmid DNA Purification System see Note 6). One microliter of resultant plasmid solution is applied to agarose gel electrophoresis to examine the yields of plasmids. 

Three to several colonies for each sample are inoculated into the same 600 pL of LB medium containing 50 pg/mL of kanamycin and cultured at 37°C overnight. For glycerol stock, 50 pL of the culture is added to 25 pL of Cell Stock Buffer in a 96-well plate. The remaining suspensions are applied to the plasmid preparation with the Wizard SV 96 Plasmid DNA Purification System see Note 6). One microliter of the resultant plasmid solution is directly applied to agarose gel electrophoresis for estimation of the size of plasmid in a form of covalently closed circular. Similarly, another 1 pL of the plasmid solution is treated with S fi/Pmel and analyzed by agarose gel electrophoresis for an insert size check. This plasmid solution set is the final product and is reserved in a freezer. 

---