Gel casting

Ereeze casting (59) is a hybrid of sHp casting, gel casting, and freeze drying in which a slurry is poured into a rigid mbber mold, frozen, and the frozen Hquid is removed by sublimation, ie, by freeze drying (see Cryogenics). 

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. 

Stellwagen, NC, Apparent Pore Size of Polyacrylamide Gels Comparison of Gels Cast and Run in Tris-acetate-EDTA and Tris-borate-EDTA Buffers, Electrophoresis 19, 1542, 1998. Stellwagen, NC Gelfi, C Righetti, PG, The Free Solution Mobility of DNA, Biopolymers 42, 687, 1997. 

Yin Y, Li S, Xia C, and Meng G. Electrochemical performance of gel-cast NiO-SDC composite anodes in low-temperature SOFCs. Electrochim. Acta 2006 51 2594-2598. 

PMDA, 20 266. See also Polymeric methylenedianiline (PMDA) PMDA-ODA, gel casting of, 20 271-272 PMDA-ODA structures, 10 214 PMDI polymeric isocyanate, 25 456, 457, 462. See also Polymeric methylene diisocyanate resins (PMDI) rigid polyurethanes from, 25 471-472 PMF/PMUF resins, 15 779 PMF resins, hardening of, 15 781 PMMA ionomer, 4DA and, 14 479 PM optimization (PMO), 15 466 PM process, 21 50 Pneumatic classification, 22 288 health and safety factors related to,... 

Gel cast between glass plates. Notches are cast in the top of the gel to receive samples. 

Protocol 5 Gel Casting (Fig. B.3A) System 3 gel apparatus from Biorad is popular and is widely used. 

Transfer the gel plates from the gel casting apparatus to the running unit. 

Other chemicals required are APS, N, N, N, N -tetramethylethylene-diamine (TEMED), riboflavin 5 -phosphate (FMN the phosphate is much more soluble than riboflavin itself), and glycerol. Recommended stock solutions are 25% (w/v) glycerol (25 g of glycerol and water to 100 mL total volume), 10% (w/v) APS (100 mg APS in 1 mL of water, freshly made before gel casting), and 0.1% (w/v) FMN (50 mg FMN in 50 mL water). TEMED is used neat. 

Allow the solution to cool for 5 min, swirling the flask gently every so often to prevent the gel from hardening. When the outside of the flask is cool enough to touch, pour the solution into an agarose-gel cast fitted with a 10-well comb at one end (consult the instructor). Allow the gel to set ( 40 min). Remove the comb, and place the gel in an agarose-gel electrophoresis chamber. Add 0.5X TBE buffer to the chamber until it completely covers the gel and fills the wells. 

Allow the flask to cool at room temperature (swirling occasionally) for 5 min. Pour the solution into a gel cast fitted with a 10-well comb at one end. Allow the agarose to set ( 40 min). 

Cheng et al. (2008) reported LaCoOs nanopowders for intermediate temperature solid oxide fuel cells prepared by an aqueous gel-casting technique at 600 °C. The performance of La-Sr-Mn-O (LSM) electrode impregnated with as-synthesized LaCoOs nanopowders showed a significant improvement. 

Millan, A., Baudin, C., Moreo, R. et al., Influence of gelling additives in the green properties of AI2O3 bodies obtained by aqueous gel casting, Key Eng. Mater., 206, 413, 2002. 

Gel casting and handling are costly, labor intensive, and not readily automated. 

Melt the agarose in a microwave and swirl to ensure even mixing. Let the melted agarose cool without gelling before pouring onto the gel-casting tray. 

After the gel has solidified, withdraw the gel comb, taking care not to tear the sample wells. Place the gel-casting tray containing the gel in the electrophoresis tank. Add IX TAE to cover the gel completely. 

Figure 11.10. Two-dimensional separation of E. coli protein mixture, using IEF with carrier ampholytes, pH range 3-10, in the first (horizontal) direction, and SDS-PAGE was mn from top to bottom in the second dimension on a 9-14% polyacrylamide gradient running gel cast with 0.1% SDS.12 [Reprinted, with permission, from P. H. O Farrell, The Journal of Biological Chemistry 250 (No. 10 May 25), 1975, 4007 4021. High Resolution Two-Dimensional Electrophoresis of Proteins . Copyright 1975 by the American Society for Biochemistry and Molecular Biology, Inc.]...

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