Acrylamide concentration

Tokita et al. [394] measured diffusion of different molecules with molecular weights varying from 18 to 342 in polyacrylamide gels with constant percentage cross-linker and varying total acrylamide concentration. They found the data to be in good agreement with the stretched exponential of the form... 

As described in the introduction, certain cosurfactants appear able to drive percolation transitions. Variations in the cosurfactant chemical potential, RT n (where is cosurfactant concentration or activity), holding other compositional features constant, provide the driving force for these percolation transitions. A water, toluene, and AOT microemulsion system using acrylamide as cosurfactant exhibited percolation type behavior for a variety of redox electron-transfer processes. The corresponding low-frequency electrical conductivity data for such a system is illustrated in Fig. 8, where the water, toluene, and AOT mole ratio (11.2 19.2 1.00) is held approximately constant, and the acrylamide concentration, is varied from 0 to 6% (w/w). At about = 1.2%, the arrow labeled in Fig. 8 indicates the onset of percolation in electrical conductivity. 

FIG. 9 Measured self-diffusion coefficients at 25°C for toluene (A), water ( ), acrylamide ( , and AOT ( ) in water, toluene, and AOT reverse microemulsions as a function of cosurfactant (acrylamide) concentration, f (wt%). The breakpoint at about 1.2% acrylamide approximately denotes, the onset of percolation in electrical conductivity. 

Acrylonitrile produced industrially via propylene ammoxidation contains trace amounts of benzene. When using Pseudonocardia thermophila JCM3095 or Rhodococcus rhodochrous J-1 as microbial NHase catalyst for conversion of acrylonitrile to acrylamide, concentrations of benzene of <4 ppm produced a significant increase in the reaction rate [16]. Maintaining the concentration of HCN and oxazole at <5 ppm and <10 ppm respectively produced high-quality acrylamide suitable for polymerization. 

Gels, 23 54. See also Aerogels Hydrogels Polymer gels Silica gels Xerogels acrylamide concentration of, 9 750 classification by body part and use, 7 842t... 

SDS-PAGE was performed essentially by the method of Laemmli and Favre (39) using 3% and 7.5% (w/v) total acrylamide concentrations in the stacking and resolving gels respectively. Sodium dodecyl sulphate was at a concentration of 0.1% (w/v). Staining for protein was accomplished using Coomassie Brilliant Blue R-250, while peroxidase activity was localized by the method of Thomas et al. (40). 

If Ry of different proteins should be compared, the calculations have to be made from the same slab gel to eliminate variances in acrylamide concentration, polymerization, and electrophoretic conditions. 

Fig. 2.1. Semilogarithmic plot of molecular weight (Mr) of marker proteins vs relative mobility (Rf) of marker proteins in gels of different acrylamide concentrations %T. Proteins 1 aprotinin (6.5 kD) 2 lysozyme (14.5 kD) 3 soybean trypsin inhibitor (21.5 kD) 4 carbonic acid anhydrase (31 kD) 5 hen ovalbumin (45 kD) 6 bovine serum albumin (66 kD) 7 phosphorylase b (97.4 kD) 8 8-galactosidase (116 kD) 9 myosin (205 kD)...

PAGE systems described in this chapter are well established nevertheless, modifications concerning acrylamide concentration (%T as well as %C) maybe done to optimize the separation conditions. 

The Laemmli SDS-PAGE protocol is one of the most important analytical techniques in analytical protein separation. It is a system with discontinuous pH gradient (disc electrophoresis) and consists of a stacking and a separation gel different in acrylamide concentration and pH. The separation gel may be formed with homogenous acrylamide concentration or with an increasing gradient. 

Table 2.1. Recipes for casting gels with homogeneous acrylamide concentration (Laemmli system)...

This system allows the separation of alkali-labile proteins (e.g., acylphosphate phosphoproteins) under denaturating conditions according to their molar mass. Despite the low acrylamide concentration (%T = 5.61, %C = 3.61), the separation force is remarkable. Because it is a SDS-containing system, the migration is from to + despite the low pH. 

Table B3.1.1 Recommended Acrylamide Concentrations for Protein Separation by SDS-PAGE...

For preparing gels choose an acrylamide concentration such that the protein will migrate as a sharp, tight band with an Rj between 0.2 and 0.8. Casting a gel in advance allows complete polymerization, reduces the amount of oxidants and free radicals, and minimizes the possibility of blocking the N terminus or other amino groups of the protein. 

Polyacrylamide is a cross-linked polymer of acrylamide. These gels are more difficult to prepare than agarose. Monomeric acrylamide (which is a known neurotoxin) is polymerized in the presence of free radicals to form polyacrylamide. The free radicals are provided by ammonium persulfate and stabilized by TEMED (/V/V/V/V -tetramethylethylenediamine). The chains of polyacrylamide are cross-linked by the addition of methylene-bisacrylamide to form a gel whose porosity is determined by the length of chains and the degree of cross-linking. The chain length is proportional to the acrylamide concentration usually between 3.5 and 20%. Cross-linking bis-acrylamide is usually added at the ratio 2 g bis/38 g acrylamide. 

The gel concentration required for polyacrylamide gel electrophoresis (PAGE) to achieve optimal resolution of two proteins (or nucleic acids) can be determined by measuring the relative mobility of each protein in a series of gels of different acrylamide concentrations to construct a Ferguson plot (log 10Rf versus %7)... 

Try to improve the resolution of the restriction fragment by altering the running conditions on the gel (e.g. longer running times, lower loading, altering the acrylamide concentration). Alternatively use a different restriction enzyme... 

Langen, H., Roder, D., Juranville, J.-F., and Fountoulakis, M. (1997). Effect of protein application mode and acrylamide concentration on the resolution of protein spots separated by two-dimensional gel electrophoresis. Electrophoresis 18, 2085-2090. 

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