Isoelectric focusing analytical

Of the various support matrices that have been used for IEF, only polyacrylamide and agarose gels have come into widespread use.40 The structural features of these two types of gels are very similar. 

Polyacrylamide gels are formed by the copolymerization of acrylamide (CH2 =CH-CO-NH2) and a cross-linking comonomer, N, N -methylene- 

During polymerization, acrylamide and bisacrylamide monomers couple together across their double bonds. Linear chains of polyacrylamide form with bisacrylamide molecules cross-linking adjacent chains. The pendant carboxamide groups (-CO-NH2) from the acrylamide monomer are subject to hydrolysis to carboxyls ( COO ) as gels age.42 Only freshly made gels should be used in most IEF work. 

The rate of polymerization is dependent on the net concentration of monomers and free radicals, the temperature, and the purity of the reagents. All of these items must be controlled for reproducible gels. Because the reaction requires free radicals, any contaminants that can act as free radical traps will inhibit polymerization. Since oxygen is the most abundant radical trap, proper degassing to remove dissolved oxygen from acrylamide solutions 

The proper choice of ampholyte range is very important to the success of an IEF fractionation. Ideally, the pH range covered by the focused carrier ampholytes should be centered on the pis of the proteins of interest. This insures that the proteins of interest focus in the linear part of the gradient with many extraneous proteins excluded from the separation zone. Carrier ampholyte concentrations of about 2% (w/v) are best. Concentrations of ampholytes below 1% (w/v) often result in unstable pH gradients. At concentrations above 3% (w/v) ampholytes are difficult to remove from gels and can interfere with protein staining. 

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Bacteria produce chromosomady and R-plasmid (resistance factor) mediated P-lactamases. The plasmid-mediated enzymes can cross interspecific and intergeneric boundaries. This transfer of resistance via plasmid transfer between strains and even species has enhanced the problems of P-lactam antibiotic resistance. Many species previously controded by P-lactam antibiotics are now resistant. The chromosomal P-lactamases are species specific, but can be broadly classified by substrate profile, sensitivity to inhibitors, analytical isoelectric focusing, immunological studies, and molecular weight deterrnination. Individual enzymes may inactivate primarily penicillins, cephalosporins, or both, and the substrate specificity predeterrnines the antibiotic resistance of the producing strain. Some P-lactamases are produced only in the presence of the P-lactam antibiotic (inducible) and others are produced continuously (constitutive). 

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

The more abundant lyase produced by FORL has been purified to homogeneity as it is shown by analytical isoelectric focusing (figure 5). The data in Table 1 show that a 32.48-fold increase in specific activity is achieved with a recovery of approximately 2.36%. The enzyme showed an "endo" type of action and a great specificity for pectin. 

Figure 1. Analytical isoelectric focusing of cellulases from Trichodtrma ree-sei. Detection of CBH I and EG I activities using MeUmbLac, in the absence (A) and presence (B) of 10 mM cellobiose. Lane 1, EG I lane 2, EG I (iso-components) lane 3, CBH I (pi 3.9 component) lane 4, EG I-CBH I mixture). Gels were flooded with the fluorogenic substrate (pH 5.0) and after 5-10 min (room temperature) photographed (Polaroid 57, green filter) on a long wavelength UV-transilluminator (8).

Figure 5. Analytical isoelectric focusing in the pH range 3.5-9.5 (Am-pholine PAG Plate, LKB-Produkter AB, Bromma, Sweden) of filtrate samples from a culture of Thermoactinomyces sp. Separation carried out for 1.5 hr at a constant power of 1 W/cm (30). Protein concentration 0.5 mg mL 1 50 pJL applied with glass filter paper (Whatman GF/A) (6).

K9. Kashyap, M. L., Srivastava, L. S., Hynd, B. A., Gartside, P. S., and Perisutti, G., Quantitation of human apolipoprotein G-III and its subspecies by radioimmunoassay and analytical isoelectric focusing Abnormal plasma triglyceride-rich lipoprotein apolipoprotein C-III subspecie concentrations in hypertriglyceridemia. J. Lipid Res. 22, 800—810 (1981). 

Purity is indicated by a clean, single, sharp peak on analytical RP-HPLC. The presence minor peaks of shoulders or a distorted peak shape, are indicators that the material needs further purification. To further confirm purity an orthogonal separation method should be used. For example, analytical ion-exchange HPLC, Fig 2E, for which the separation is based on ionic rather than nonpolar interactions. Analytical isoelectric focusing can also be used. 

Analytical isoelectric focusing was carried out using a Pharmacia-LKB Multiphor II unit with a 4.0-6.5 PAG plate. After running the gel, it was sliced in 1 cm pieces and the slices were incubated overnight at 30°C with 25.0 mM DKP in 25.0 mM potassium phosphate, pH 8.0, containing 1.25 mM EGTA. Samples were analyzed by TLC for the production of a-AP which was then correlated with the pH in the gel, measured by a flat electrode. 

Figure 6. pi determination using a flat bed analytical isoelectric focusing gel. pi was determined to be between 4.2 and 4.7. Points ( ) were actual pH measurements while the line is a linear least squares fit. 

Figure 1. Analytical Isoelectric Focusing at Various Stages in the Purification of Human Cathepsin D. (a) Homogenate,...

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