Isoelectric focusing polyacrylamide gels

See also-. Capillary Electrophoresis Overview. Electrophoresis Principles Isotachophoresis Isoelectric Focusing Polyacrylamide Gels Two-Dimensional Gels Blotting Techniques. 

See also Electrophoresis Overview Principles Iso-tachophoresis Isoelectric Focusing Polyacrylamide Gels Blotting Techniques Clinical Applications. 

See also Blood and Plasma. Capillary Electrophoresis Overview. Cerebrospinal Fluid. Clinical Analysis Sample Handling. Electrophoresis Principles Isoelectric Focusing Polyacrylamide Gels Clinical Applications. Immunoassays Overview. Immunoassays,... 

See also-. Electrophoresis Isoelectric Focusing Polyacrylamide Gels Two-Dimensional Gels Proteins. Liquid Chromatography Liquid Chromatography-Mass Spectrometry Biotechnology Applications. Mass Spectrometry Ionization Methods Overview Electrospray Matrix-Assisted Laser Desorption/lonization Multidimensional Peptides and Proteins. Proteins Traditional Methods of Sequence Determination. 

Hoffmeister, H., Allen, R. C., and Maurer, H. R. (1974). Electrophor. Isoelectric Focusing Polyacrylamide Gel, [Proc. Small Conf.] 266. 

Rodbard, D Chrambach, A, In Electrophoresis and Isoelectric Focusing in Polyacrylamide Gels Allen, RC Mauer, HR, eds. Walter E Gruyter New York, 1974 28. 

Ultrathin-layer isoelectric focusing in polyacrylamide gels on polyester films was performed as described (Radola, 1980). Polygalacturonase activity was detected by the print technique with a dyed substrate (Ostazin Brilliant Red-D-galacturonan DP 10) (Markovic et al., 1992) or by the print technique with colouress D-galacturonan DP 10 dyed additionally with ruthenium red (Sigma, Germany). 

Innovations in separation science continued on this theme and provided one of the most powerful separation techniques used in biochemistry, where proteins are separated with isoelectric focusing (IEF) applied in one direction, and gel electrophoresis (GE) applied at aright angle to the first separation direction (O Farrell, 1975 Celis and Bravo, 1984). In this case, proteins are first separated according to their isoelectric point, measured in p/units, and then according to their molecular weight by gel electrophoresis. The size separation step is usually aided by addition of a surfactant, most typically sodium dodecyl sulfate (SDS), and the gel material is a polyacrylamide formulation. 

First Dimension Optimization After the second-dimension separation has been developed, the first-dimension flow rate is determined. This includes selecting a first-dimension column diameter to work at the flow rate selected. We illustrate the selection process with an application that addresses a column method for proteins that functions as a replacement for planar 2D gel electrophoresis (2DGE) within a narrow molecular weight and p/range. In the planar experiment, isoelectric focusing is performed in the first dimension and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS/PAGE) in the second dimension. 

The following procedure relates to electrophoretic protocols where the first dimension is developed by isoelectric focusing (in tube gels) and the second dimension is a size exclusion separation by SDS polyacrylamide electrophoresis in a slab gel. 

CaM was purified from porcine brain. The purity of proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. CaM and PDE were cross-linked with l-ethyl-3(3-dimethylamino-propyl) carbodiimide (EDC) or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) in a buffer solution of 0.1 M HEPES (pH 7.1) in the presence of 1 mM CaCl2. After a buffer solution containing 2 mM EGTA added into the reaction solution, the CaM-PDE hybrid was separated from other ingredients by gel chromatography on a Sepharose CL-6B solumn. 

A, Absorption chi, chlorophyll car, carotenoid EET, excitonic energy transfer EF, exoplasmic fracture face EM, electron microscopy FWHM, full width at half maximum lEF, Isoelectric Focusing, LD, linear dichroism LHC, light harvesting complex PAGE, polyacrylamide gel electophoresis PF, protoplasmic fracture face PS, photosystem RC, reaction centre SDS, sodium dodecyl sulphate SSTT, single step transfer time. 

Fig. 1.43 Strategies for protein identification. (A) 2D gel electrophoresis approach. (B) 2D liquid chromatography approach. lEF Isoelectric focusing, sex strong cation exchange column, RP reverse phase column, SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Electrophoretic Methods Systems for polyacrylamide gel electrophoresis, 104, 237 preparative isoelectric focusing,104, 256 gel... 

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