Polyacrylamide gels sample preparation

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. 

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. 

If the supporting medium is a sheet or membrane, it is soaked in the buffered electrolyte, the excess being removed before the sample is applied. Alternatively a gel, such as agar or polyacrylamide, can be prepared in the buffer and a slab or column used for the separation process. 

Craven BA, Totty N, Harnden P, Selby PJ, Banks RE. (2002) Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis evaluation of tissue preparation and sample limitations. Am J Pathol 160, 815-22. 

Slab gels are now mote widely used than column gels. A slab gel on which several samples may be analyzed is more convenient to make and use than several individual column gels. Slab gels also offer the advantage that all samples are analyzed in a matrix, environment that is identical in composition. A typical vertical slab gel apparatus is shown in Figure 4.3. The polyacrylamide slab is prepared between two glass plates that are separated by... 

Obtain a precast SDS-polyacrylamide slab gel or prepare one according to instructions in Experiment 4. The recommended gel is 12°/o acrylamide with a thickness of 0.75 mm. Protein samples are prepared as follows Purified proteins (transferrin, bovine serum albumin, a, -antitrypsin, a-lactalbumin from Experiment 4, and molecular weight standards) are supplied in Tris buffer, pH 6.8 solutions at a concentration of 1 mg/mL. Sera samples have been diluted and are ready for use. Prepare protein samples for electrophoresis in 0.5-mL microcentrifuge tubes with attached caps. Label the tubes from 1 to 5 as below or per your Instructor s directions. 

Fig. 2. SDS-PAGE of an antibody-toxin conjugate preparation. (A) Antibody (starting material). (B) Abrin A chain conjugate (pooled fractions shown in Fig. 1). Samples were prepared under nonreducing conditions and run on a 2—27% gradient polyacrylamide gel.

The protocol involves a classical SDS-PAGE (10% polyacrylamide) run, followed by transfer onto a Western blot membrane and immunodetection with an anti-pIII antibody. Nevertheless, special care must be taken during sample preparation, because phages are very stable and difficult to denature. The protocol is similar to typical SDS-PAGE sample preparation, except that / -mer cap toe thanol should be replaced by fresh dithiothreitol (DTT, 5 mM final concentration), and the samples should be boiled in a water bath for at least 15 min. Moreover, because the pIII-fusion protein is a minor component of the virion, a large amount of phages should be loaded onto the gel, typically around 1012 phages per lane. 

When the folding reactions have reached equilibrium, 2 fiL of each sample is loaded into separate lanes of a native 8% polyacrylamide gel that was prepared and prerun as described above (< 10 ° C). It is helpful to use narrow tips that allow the sample to be placed near the bottom of the well. The current should be applied to the gel as soon as possible after the samples are loaded. Gels must be run long enough to resolve the conformational species of interest, but not so long that smaller RNAs run off the bottom of the gel. To resolve the folded and unfolded forms of the Tetrahymena ribozyme, gels are run 4 h at 15 W. 

Samples are prepared and electrophoresed as described in Protocol 1 except mini SDS-polyacrylamide gels are used that measure approximately 5.5 cm X 9 cm X 0.75 mm. Suitable gels are supplied by Bio-Rad for use with the Mini Protean II electrophoresis apparatus. 

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