Polyacrylamide-gel

Polyacrylamide is a well-defined, stable, and rather inert gel with a pore size that can be easily varied. It is mechanically strong, easily handled, and transparent. Due to relatively small pore size, the eddy diffusion is low and hence contributes little to band broadening. However, due to the small pore size, polyacrylamide gels are less suitable for separation of very large molecules such as proteins with molecular mass 300 000 (300 kDa). 

Polyacrylamide gels are made by polymerization of acrylamide (toxic) with N, -methylenebisacrylamide in the presence of free radicals generated from either ammonium persulfate (oxidative chemical initiator) or riboflavin-5 -phosphate (photochemical initiator). The reaction is controlled by equimolar concentrations of N,N,N, N -tetramethylethylene diamine (TEMED) as catalyst. 

The pore size is characterized by %T (total acrylamide concentration) and %C  

In polyacrylamide gel separations, a discontinuity in the gel and/or electrolyte composition (pH) can give rise to a concentration of the start band prior to the separation, giving better efficiency and hence resolution. 

When the tracking dye has migrated to the bottom of the column, the gel is removed using pressure, and proteins are fixed in place by precipitation [this occurs during a soaking step in a solution of trichloroacetic acid (TCA)] and are then stained for detection. 

Slab PAGE allows simultaneous electrophoresis of a number of samples to be performed under identical conditions. Slab PAGE has a higher resolving power than column PAGE, and is often used for molecular weight and purity determinations. Because the slab is relatively thin, heat dissipation is more efficient in a slab than in a column. The slab can be used vertically or horizontally, but in practice, the horizontal slab method is used only when the monomer plus cross-linker concentrations are low and the gel is soft. A slab gel is prepared in the same way as a column gel, except that a comb, or slot-former, is inserted before polymerization. Removal of the comb after the gel has set leaves sample wells that are separated from each other by continuous strips of gel. 

The pore size of a polyacrylamide gel controls the mobility and resolution of components because of the sieving effect of the pores on macromolecular species.6 The pore size may be controlled by varying the total concentrations of monomer and cross-linker, and by varying their ratio. Gel compositions are dehned by two parameters, their %T and %C values, that represent the total and crosslinker contents, respectively. These parameters are defined by Eqs. 9.5 and 9.6. 

The molecular weight of a protein and its net charge may be determined by performing electrophoresis on gels with constant %C, by varying %T from gel to gel. After separation, the migration of the protein is measured relative to a tracking dye, and the Rf value is calculated  

Rf = (migration distance of protein)/(migration distance of marker) (9.7) 

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Electrophoresis is used primarily to analyze mix tures of peptides and proteins rather than individual ammo acids but analogous principles apply Because they incorporate different numbers of ammo acids and because their side chains are different two pep tides will have slightly different acid-base properties and slightly different net charges at a particular pH Thus their mobilities m an electric field will be differ ent and electrophoresis can be used to separate them The medium used to separate peptides and proteins is typically a polyacrylamide gel leading to the term gel electrophoresis for this technique... 

The contents of each tube are then subjected to electrophoresis m separate lanes on the same sheet of polyacrylamide gel and the DNAs located by autoradiography A typical electrophoresis gel of a DNA fragment containing 50 nucleotides will exhibit a pattern of 50 bands distributed among the four lanes with no overlaps Each band cor responds to a polynucleotide that is one nucleotide longer than the one that precedes it (which may be m a different lane) One then simply reads the nucleotide sequence according to the lane m which each succeeding band appears... 

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. 

Polyacrylamide gel Polyacrylamide gels Polyacrylamide grout Polyacrylamide pAM) P o ly acryl arm d es... 

Size Isomers. In solution, hGH is a mixture of monomer, dimer, and higher molecular weight oligomers. Furthermore, there are aggregated forms of hGH found in both the pituitary and in the circulation (16,17). The dimeric forms of hGH have been the most carefully studied and there appear to be at least three distinct types of dimer a disulfide dimer connected through interchain disulfide bonds (8) a covalent or irreversible dimer that is detected on sodium dodecylsulfate- (SDS-)polyacrylamide gels (see Electroseparations, Electrophoresis) and is not a disulfide dimer (19,20) and a noncovalent dimer which is easily dissociated into monomeric hGH by treatment with agents that dismpt hydrophobic interactions in proteins (21). In addition, hGH forms a dimeric complex with ( 2). Scatchard analysis has revealed that two ions associate per hGH dimer in a cooperative... 

Alcosorb 400 Water-Retaining Polymer," undated product Hterature, AHied CoUoids describes agricultural uses of cross-linked polyacrylamide gels. 

Fig. 3. Sodium dodecyl sulfate—polyacrylamide gel electrophoretic pattern for molecular weight standards (lane 1) water-extractable proteins of defatted soybean meal (lane 2) purified IIS (glycinin) (lane 3) and purified 7S (P-conglycinin) (lane 4) where the numbers represent mol wt x 10. The gel was mn in the presence of 2-mercaptoethanol, resulting in the cleavage of the disulfide bond linking the acidic (A bands) and basic (B bands) polypeptides of the...

Anhydrotetracycline oxygenase from Streptomjces aureofaciens which cataly2es the conversion of anhydrotetracycline to dehydrotetracycline, has been isolated and characterized as a flavin-dependent oxygenase (83). It consists of two subunits of mol wt = 57, 500 based on SDS/polyacrylamide—gel electrophoresis. The cosynthetic factor 1 of Streptomjces aureofaciens involved in the reduction of 5a,lla-dehydrochlortetracycline to chlortetracycline, has been identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin. This work was aided by comparison of spectral data with that of an authentic sample obtained from the hydrolysis of coenzyme F-420 (84). 

Disc electrophoresis was first iatroduced ia the early 1960s (11—13) as various techniques using polyacrylamide gels were being explored and designed. Original work employed several buffer systems and different polyacrylamide gels in order to first concentrate and then separate compounds (14). 

Polyacrylamide Electrophoresis. Polyacrylamide gels are synthesized through the combination of acrylamide [79-60-1] (qv), CH2=CHC0NH2, monomer and a cross-linking comonomer (see Acrylamide POLYMERS). Typically, the cross-linking comonomer of choice is... 

N,]S2-diaHyltartardiamide (DATD) [58477-85-3] (37). The cross-linking of polymerized monomer with the comonomer is what controls the pore size of the gel polymer mesh. This level of pore size control makes polyacrylamide gel electrophoresis an effective analytical tool. 

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. 

Polyacrylamide gel electrophoresis is one of the most commonly used electrophoretic methods. AnalyMcal uses of this technique center around protein characterization, for example, purity, size, or molecular weight, and composition of a protein. Polyacrylamide gels can be used in both reduced and nonreduced systems as weU as in combination with discontinuous and ief systems (39). 

An example of the use of polyacrylamide gels in an ief system in combination with immun obi otting is given in Reference 40 where this method is used to detect low quantities of group specific component (GC) subtypes. Another example of polyacrylamide in combination with ief is given in Reference 41 where the technique is used as a screening tool for inheritance of a certain polymorphic protein. 

Amido black is a commonly used stain, but it is not very sensitive. It is often used to visualize concentrated proteins or components that are readily accessible to dyes such as proteins that have been transferred from a gel to nitrocellulose paper. Two of the more sensitive and more frequently used stains are Coomassie Brilliant Blue (R250 and G250) and silver stains. Because these stains interact differently with a variety of protein molecules, optimization of the fixative and staining solutions is necessary. The Coomassie stains are approximately five times more sensitive than amido black and are appropriate for both agarose and polyacrylamide gels. The silver stain is approximately 100 times more sensitive than Coomassie and is typically used for polyacrylamide gels. 

Plus One REPEL-SILANE ES (a solution of 2% w/v of dichloromethyl silane in octamethyl cyclooctasilane) is used to inhibit the sticking of polyacrylamide gels, agarose gels and nucleic acids to glass surfaces and is available commercially (Amersham Biosciences). 

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