SDS-PAGE method

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

Discontinuous zonal electrophoresis, known as disc-electrophoresis for short, is the electrophoretic technique that is most often used in protein analysis. The method, originally developed by Omstein and Davis, has given rise to several derivative techniques, notably the well-known SDS-PAGE method of Laemmli (1970). The technique is thoroughly covered in the literature, reflecting its importance in protein... 

The SDS-PAGE method of Laemmli (1970) is convenient to verify the purity of the Ig, particularly with the very sensitive silver staining method (Section 16.1.1). It is good practice to analyse both unreduced and reduced (with 2-ME) samples. 

Since its original introduction (Laemmli 1970) this combination of polyacrylamide gel electrophoresis (PAGE) with denaturation by SDS has become well established but further improvements to the SDS-PAGE method are still being developed (e.g., Schagger 1987). Note that getting rid of the SDS detergent is essential before LC—MS/MS analysis is attempted, since the dodecylsulfate anions form ion pairs with the protonated peptides and severely reduce the electrospray response of the latter. 

Several electophoretic methods (SDS-PAGE, native PAGE and lEF) showed that fraction Q2 was almost pure, some faint contaminating protein bands were found (Fig. 9). The major band upon SDS-PAGE was at 42 kDa. lEF indicated an isoelectric point at pH 4.3 for the most prominent band... 

Products were analysed by SDS-PAGE followed by Western blotting (Burnette 1981). RGase antibody production is described by van der Veen et al. 1991 This method can be used to determine the presence of RGase in different enzyme-preparations and culture-filtrates. 

SDS-PAGE was performed by the method of Laemmli [17]. The methods for native PAGE, isoelectric focussing, detection of esterase activity in electrophoresis gels, and assays for protein glycosylation have been described elsewhere [5]. 

PGE was isolated as desribed in Material and Methods. SDS-PAGE electrophoresis of purified protein showed a single band migrating at approximately 60 kDa. This observation is not in the agreement with the calculated molecular weight of 35 584. However a similar effect has been observed previously in case of PGI and PGC. Apart of the N-glycosylation which plays role in all PGs (Fig. 3), O-glycosylation may also be present as indicated by the band size shift after a treatment of PGE with O.IM NaOH (data not shown). 

SDS-PAGE was performed by the method of Laemmli (17), with 7.5% polyacrylamide. 

The specifications and standardization include raw materials, preparation method of the standard solution, concentration of proteins, and the main band on SDS-PAGE. The outline of the procedure for preparation of the calibrators is shovm in Eig. 4.2. Table 4.5 shows the raw materials and the preparation method of the initial extract. To prepare the calibrators, the raw materials are extracted by the standard solution containing SDS and mercaptoethanol. The initial extract is prepared by centrifugation and filtration of the extract. The diluted extract is then prepared by 10-fold dilution of the initial extract with phosphate-buffered saline (PBS pH 7.4). The protein concentration of the diluted extract is assayed using the 2-D Quant kit (Amersham Bio Sciences). The standard solution is then... 

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

The utility of MALDI-FTMS analysis for use in chemotaxonomic applications has been established, but this method can be applied to other areas of interest, such as biomedical and environmental analyses. A common method used by biochemists and biologists today is recombinant overexpression of proteins using bacterial whole cells in cases where large quantity of a protein is desired. The main method presently used to determine if the overexpression was successful is the use of SDS-PAGE (sodium dodecylsulfate-poly acrylamide gel... 

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. 

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