Polyacrylamide gels immobilization

Immobilization on attachment surface - Work on the use of polyacrylamide gel-immobilized microarrays for nucleic acids has been reported by Zlatanova and Mirzabekov [25]. The use of unmodified oligos to fabricate DNA microarrays where acid-washed slides are coated with epoxy silane before probe deposition has also been reported [26]. 

Figure 3.7 Rotating stabilized cell (RSC) technique a polyacrylamide gel immobilizes the aqueous phase.

Mifflin and associates described a membrane electrode for the quantitative analysis of penicillin in which the enzyme penicillinase is immobilized in a polyacrylamide gel that is coated on a glass pH electrode. The following data were collected for a series of penicillin standards. 

Chibata, I., Immobilized Microbial Cells with Polyacrylamide Gel, Carrageenan and their Indushial Application, In Immobilized Microbial Cells , chap. 3. American Chemical Society, Washington, D.C., 1979. 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

Except the physical entrapment of horseradish peroxidase in a polyacrylamide gel used by Freeman and Seitz23, immobilization of the... 

Adenosine triphosphate (ATP) is one of the most important cofactors involved in many of the synthetic reactions going on within the cell. Its recent large scale in vitro enzymatic synthesis from adenosine and acetylphosphate is of particular interest. Three enzymes immobilized in polyacrylamide gel were used adenosine kinase, adenylate kinase and acetate kinase (lip. ... 

Tucker MD, Barton LL, Thomson BM. 1998. Reduction of Cr, Mo, Se, and U by Desulfovibrio desulfuricans immobilized in polyacrylamide gels. J Ind Microbiol Biotechnol 20 13-19. 

Figure 3 Biosynthesis and purification of 90-kD elastin analogue analyzed by denaturing polyacrylamide gel electrophoresis (10-15% gradient, visualized by silver staining). Lanes 1-7 time course of target protein expression at 0, 30, 60, 90, 120, 150, and 180 minutes after induction. Lane 9 soluble lysate of induced E. coli expression strain BLR(DE3)pRAMl. Lanes 10-13 protein fractions obtained from immobilized metal affinity chromatography of the lysate on nickel-NTA agarose (imidazole gradient elution). Lanes 8,14 protein molecular weight standards of 50, 75, 100, and 150 kD.

Fig. 15 Chemistry of immobilization of OND probes into polyacrylamide gel pads...

Brandley and Schnaar [140] immobilized a synthetic nonapeptide, Tyr- Ala-Val Thr-Gly Arg-Gly-Asp-Ser, on a polyacrylamide gel, which had been prepared by a ternary copolymerization of acrylamide, bisacrylamide and the acrylic ester of. V-hydroxysuccinimide. They reported that Balb/c 3T3 mouse fibroblast cells (in Hepes-buffered Dulbecco s modified Eagle medium) adhered readily to the peptide-derivatized surfaces, even in the absence of serum, although long-term cell growth required the presence of serum. It was noticed that reference nonapeptide. Tyr-Arg-Leu-Glu-Asp-Pro-Ala-Met-Trp, which has no RGD sequence, failed to promote cell-attachment. 

Immobilized enzymes are becoming increasingly important in commercial processes. In this experiment, students will trap molecules of the enzyme horseradish peroxidase within a polyacrylamide gel matrix. The reaction kinetics and thermal stability of the immobilized enzyme will be measured. This experiment introduces students to the use of enzymes in biotechnology. 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Similar results were obtained when polyethylene was modified by grafting acrylamide and cholesterol ester of N-methacryloyl-[3-alanine to get 0.3 + 0.1 mg UChD/ cm2. The efficiency of UChD bound to polymer was close to that of UChD immobilized in a polyacrylamide gel the graft copolymers sorbed from the plasma solution the amount of 0.9 mg heparin/mg of immobilized UChD. The clotting time of blood contacting the modified (but not heparinized) polyethylene was 6 min, whereas on its heparinization the time increased to 30 minutes. The latter figure was actually unaltered after sixfold repetition of the heparin sorption, subsequent washing heparin off the polymer and its repeated sorption. 

An unusual type of derivative is the complex that forms between urease and bentonite in acid medium (61). The adsorbed form was found catalytically active. Similarly, urease immobilized in a polyacrylamide gel matrix has been used to prepare a urea-specific enzyme electrode (62). Yet another active water-insoluble derivative has been prepared (63) by allowing p-chloromercuribenzoate-treated urease to react with a diazotized copolymer of p-amino-D,L-Phe and L-Leu. Urease has been found to retain about 20% of its original activity when encapsulated in 100 n microcapsules of benzalkonium-heparin in collodion (64). 

Colloidal platinum dispersions, prepated by photoreduction of tetrachloroplatina-te(II) ion in the presence of a copolymer of IV-vinyl-2-pyrrolidone and acrylamide, are treated with polyacrylamide gel having amino groups, resulting in stable immobilization of colloidal particles onto the gel. The immobilized catalysts exhibit high activities for the hydrogenation of olefins at 30 °C and 1 atm 87). 

C. butyricum was immobilized in polyacrylamide gel membrane and the immobilized whole cells were fixed on the anode. A linear relationship was obtained between the steady-state current and the BOD from 0 to 250 ppm. The steady-state current was reproducible within 7 % of relative error, when the standard solution (50 mg 1 glucose, 50 mg l glutamate) was measured repeatedly. The standard deviation was 2 ppm. 

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