Gel electrophoresis SDS-PAGE

SDS/PAGE gel electrophoresis system (Bio-Rad, Novex, San Diego, CA or equivalent) supplied with gradient gels of 4-20% or homogeneous gels of 20% (see Note 2). 

Vincenzi, S. and Curioni, A. (2005) Anomalous electrophoretic behaviour of a chiti-nase isoform from grape berries and wine in glycol chitin-containing SDS-PAGE gels, Electrophoresis, 26, 60-63. 

SDS-)PAGE (gel electrophoresis) Molecular size, aggregation and type of aggregates (noncovalent, cystein-bonded) Simple, potentially high resolution... 

Check the purity of the purified protein with native and SDS/ PAGE gel electrophoresis with silver staining detection (Fig. 2a), followed by Western-blot and immunodetection of the product (Fig. 2b). 

Check purity of SR-proteins by SDS-PAGE gel electrophoresis either followed by Coomassie staining or by immunoblotting using mAb 104 antibody.0... 

Before SDS-PAGE gel electrophoresis, cell lysates are sonicated to break DNA and reduce viscosity. Samples are heated at 70°C for 10 min, centrifuged at the maximal speed in a microcentrifiige for 5 min, and then loaded and separated by SDS-PAGE. 

Most of our work has focused on the MUPs of Mus domesticus. Our initial surveys aimed to assess the molecular heterogeneity of these proteins in inbred laboratory mouse strains. The group of MUPs synthesised and excreted by any one strain was very similar, indistinguishable by simple one dimensional SDS-PAGE gel electrophoresis, a technique that discriminates proteins by virtue of their size. Previous work (Finlayson, Potter ... 

X-ray ciystallographic studies of a given protein have conclusively proved that the protein consists of four peptide chadns in association with each other. Yet SDS-PAGE gel electrophoresis of the protein gives just one band. How many explanations can you provide to justify the above observation ... 

The purity of the AE fraction was investigated by SDS-PAGE using Pharmacia PhastSystem with 10 - 15% SDS-gradient gels. Electrophoresis and silver staining of the proteins were performed as described by the manuals from Pharmacia. For determination of pi lEF 3-9 PhastSystem gels were used. 

Figure 2.1. Schematic illustration oftwo-dimensional gel electrophoresis. Proteins are extracted from the organism of interest and solubilized. The first dimension separates proteins based on isoelectric point. The pi strip is reduced and alkylated and applied to an SDS-PAGE gel for separation by molecular weight. Proteins canbe visualized using a number of staining techniques.

Sweedler reported a two-dimensional separation, where fluorescein thiocarbamyl derivatives of peptides were separated by capillary zone electrophoresis in the first dimension (Liu and Sweedler, 1996). The outlet of the capillary was wiped across the top of an SDS-PAGE gel, where peptides were then separated based on their size. 

SDS polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used analytical technique in the assessment of final product purity (Figure 7.1). This technique is well established and easy to perform. It provides high-resolution separation of polypeptides on the basis of their molecular mass. Bands containing as little as 100 ng of protein can be visualized by staining the gel with dyes such as Coomassie blue. Subsequent gel analysis by scanning laser densitometry allows quantitative determination of the protein content of each band (thus allowing quantification of protein impurities in the product). 

Urinary proteins were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), and a 70-kDa protein was identified as the major component of cat urine (Fig. 4.1 A). Comparative analysis of urinary proteins in several other mammals such as humans, mice, dogs, and cattle did not detect a 70-kDa protein. Therefore, the 70-kDa protein was purified from cat urine and characterized by biochemical methods (Miyazaki, Kamiie, Soeta, Taira and Yamashita 2003). Analysis of tissue distribution indicated that the 70-kDa protein is expressed in the kidney in a tissue-specific manner and secreted from the proximal straight tubular cells of the kidney into the urine (Fig. 4.IB). A full-length cDNA for a 70-kDa protein was cloned from a cat kidney cDNA library. The cDNA clone encoded a polypeptide of 545 amino acid residues. The deduced amino acid sequence shared 47% identity with cat carboxylesterase (CES, EC 3.1.1.1), and contained both the CES family protein motif (EDCLY) and a conserved active site motif (GESAG) associated with... 

For far Western blotting, native or recombinant proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to the nitrocellulose membranes and the blotted proteins are... 

SEC purified -D-g ucosidase. This enzyme grade was prepared by applying diafiltered SP188 to the Sephacryl S-200 column. The column was operated at a flow rate of 15 mL/min in 10 mM phosphate buffer pH 6.5 with 100 mM NaQ. -D-glucosidase activity was found in an early eluting peak which proved to be approximately 92% -D-glucosidase by SDS-polyacrylamide gel electrophoresis (PAGE)(data not shown). 

The most commonly used procedure for checking the purity of proteins is sodium do-decyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In electrophoresis, molecules move in an electrical field (see p.276). Normally, the speed of their movement depends on three factors—their size, their shape, and their electrical charge. 

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