Polyacrylamide gels, dissolving

Polyacrylamide gels were dissolved in 30% peroxide solution and added to a scintillation mixture in 1 1 2-ethoxy ethanol-toluene. After counting, the mixtures were bulked and evaporated intermittently with heat dining a 4 week period, and the accumulated peroxidised residues eventually exploded violently [1], A subsequent comment indicated that peroxidised materials may not necessarily have been formed, because solutions of organic materials in aqueous peroxide are themselves potentially explosive [2],... 

Visual examination of crystals using a light microscope does not indicate whether the crystals consist of only the protein or the protein-DNA complex. Therefore, the crystals are washed free of any uncrystallized DNA and protein several times with a solution containing the precipitant and any additives, etc. at the concentration and pH used for growing crystals (mother liquor). Finally, the crystals are separated from the mother liquor by microcentrifugation, dissolved in a suitable buffer, and analysed biochemically. The protein content is determined by SDS polyacrylamide gel electrophoresis, the protein concentration by BIO-RAD assay, and amino acid composition by mass-spectroscopy. The DNA can be detected by staining the gel with ethidium bromide or methylene blue (Jordan et al., 1985), whereas... 

The procedures described in this unit all utilize a polyacrylamide gel matrix. The gel forms when a dissolved mixture of acrylamide and bisacrylamide cross-linker monomers polymerizes into long chains that are covalently cross-linked. The gel structure is held together... 

Several of the synthetic detergents used for dissolving membranes and solubilizing integral membrane proteins. Triton X-100 and octylglucoside are nonionic detergents cetyltrimethylammonium bromide and sodium dodecylsulfate (SDS) are ionic. SDS is also an effective denaturant of proteins and is used in polyacrylamide-gel electrophoresis (see chapter 6). 

In this experiment the Pstl fragment was first digested with DNAase II in sodium acetate buffer, pH 4.7 at room temperature and the reaction halted by chilling and extraction with phenol. After precipitation the DNA was electrophoresed on an 8% polyacrylamide gel and a slice of gel, corresponding to fragments of chain length 150-250 nucleotides cut out and eluted. The 3 -terminal phosphates were removed by treatment with alkaline phosphatase and, after denaturation and removal of the phosphatase with phenol, the DNA was reprecipitated and dissolved in a small volume of water. 

Electrophoretic Separation. The net charge on a particular protein varies with the pH of the medium in which it is dissolved. Accordingly, application of an electric field to a buffered, heterogeneous protein solution often results in their differential migration in free solution or in heterogeneous systems with an inert supporting material, such as a polyacrylamide gel. Because of the difficulty of scaling up electrophoretic procedures, they are more commonly used for analytical applications than for the preparative scale purification of proteins. The use of electrophoresis is discussed in more detail in Experiment 4. 

The first commercial production of L-aspartic acid was started in 1973 by the Tanaba Seiyaku Company, Japan. The process uses aspartase contained in whole microorganisms and involves the immobilization of E. coli on polyacrylamide gel or carrageenan. The immobilized cells are then subjected to treatment in order to increase cell permeability. The substrate, fumaric acid, is dissolved in a 25 % ammonia solution and the resulting ammonium fumarate is then passed through the reactor containing the immobilized E. coli. The reaction is exothermic and the reactor has to be designed to remove the heat produced. The conversion of fumaric acid to aspartic acid is more economical than the direct fermentation of sugars. The key to economical production of L-aspartic acid for expanded use is a cheaper and more abundant source of fumaric acid. 

Figure 6. Polyacrylamide gel electrophoresis of abalone sperm and AV contents. Lanes A and E are standard proteins of known molecular mass. Lane B, whole sperm dissolved in SDS. Lane C, the acrosome vesicle content released to seawater when exocytosis of the sperm is induced by high calcium ion concentration. Lane D, purified 16-kDa lysin. (from Lewis et al., 1982).

The polyacrylamide gels were prepared by a standard redox reaction with ammonium persulfate and tetramethylethylenediamine (TEMED). Recrystallized acrylamide monomer (5 g), N,N -methylenebis(acrylamide) (0.133 g), ammonium persulfate (40 mg), and TEMED (400 xL) were dissolved in water to make a total volume of 100 mL. After thorough mixing, the preparation was poured through a small aperture into cylindrical or spherical glass molds. Upon gelation, the molds were broken and the gel samples were transferred into a cell containing an excess of water. This time was taken to be zero if = 0) for the kinetics experiments. 

Polyacrylamide gel isoelectric focusing of MDPF-labeled peptides has also been demonstrated (Stein, 1977). After the fluorogenic reaction the sample is desalted either with a molecular seiving column, such as Sephadex G-10, or by taking it to dryness and then dissolving the relatively hydro-phobic peptide fluorophor in an organic solvent. 

The polyacrylamide gels were prepared by free-radical polymerization [3]. Acrylamide, the linear constituent N,N -methylenebisacrylamide, the tetrafunctional cross-linking constituent and ammonium persulfate and N,N,N, N -tetramethylethylenediamine (TEMED), the initiators, were dissolved in water. Micropipettes with a well-defined diameter (1.4 mm) were immersed in this solution. Within 5 min, the solution gelled. After an hour, the gels were removed from the micropipettes and immersed in water to wash away residual monomers. The gels then underwent hydrolysis in a 1.2% solution of TEMED (pH 12) for more than a month. Approximately 20% of the acrylamide groups were converted to acrylic acid groups, some of which were ionized in water. 

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