SDS polyacrylamide

Size Isomers. In solution, hGH is a mixture of monomer, dimer, and higher molecular weight oligomers. Furthermore, there are aggregated forms of hGH found in both the pituitary and in the circulation (16,17). The dimeric forms of hGH have been the most carefully studied and there appear to be at least three distinct types of dimer a disulfide dimer connected through interchain disulfide bonds (8) a covalent or irreversible dimer that is detected on sodium dodecylsulfate- (SDS-)polyacrylamide gels (see Electroseparations, Electrophoresis) and is not a disulfide dimer (19,20) and a noncovalent dimer which is easily dissociated into monomeric hGH by treatment with agents that dismpt hydrophobic interactions in proteins (21). In addition, hGH forms a dimeric complex with ( 2). Scatchard analysis has revealed that two ions associate per hGH dimer in a cooperative... 

Anhydrotetracycline oxygenase from Streptomjces aureofaciens which cataly2es the conversion of anhydrotetracycline to dehydrotetracycline, has been isolated and characterized as a flavin-dependent oxygenase (83). It consists of two subunits of mol wt = 57, 500 based on SDS/polyacrylamide—gel electrophoresis. The cosynthetic factor 1 of Streptomjces aureofaciens involved in the reduction of 5a,lla-dehydrochlortetracycline to chlortetracycline, has been identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin. This work was aided by comparison of spectral data with that of an authentic sample obtained from the hydrolysis of coenzyme F-420 (84). 

Molecular weight determination The molecular weight of the purified enzyme was determined by both gel filtration chromatograph and SDS polyacrylamide gel electrophoresis (16). Toyopearl 55HW gel (Toyo SF 160K, Toyo, Co.,Ltd., Tokyo) was used for gel filtration and 0.05 N acetate buffer pH 5.25 was used as buffer. 

Figure 5 SDS Polyacrylamide Gel Electrophoresis of pectinase from different steps of purification A (1,6) standard protein (2) crude enzyme (3)...

The purified L-type channels from skeletal muscle appear to be a multisubunit protein containing the products of four different and unrelated genes. A cartoon depicting some of the features of the purified protein is shown in Fig. 1. The subunits of the channel are commonly referred to as the ai, a2, ]8, y, and 8. These have been observed to migrate in a variety of SDS-polyacrylamide gels with apparent... 

Bode, H. J., SDS-polyethyleneglycol electrophoresis a possible alternative to SDS-polyacrylamide gel electrophoresis, FEBS Lett., 65, 56, 1976. 

Takagi, T., Tsujii, K., Shirahama, K. (1975). Binding isotherms of sodium dodecyl sulfate to protein polypeptides with special reference to SDS-polyacrylamide gel electrophoresis. J. Biochem. (Tokyo) 77, 939-947. 

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. 

Fig. 8.4 Coomassie-stained SDS-polyacrylamide gel showing chloroplast transgenic lines expressing IFNa2b. Lanes 1 and 2 Total soluble protein (TSP) Lanes PH, 3 and 4 Total protein (TP).

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). 

When labeled polypeptides traveling down the axon are analyzed by SDS polyacrylamide gel electrophoresis, materials traveling in the axon can be grouped into five distinct rate components [6], Each rate component is characterized by a unique set of polypeptides moving coherently down the axon (Fig. 28-3). As specific polypeptides associated with each rate class were identified, most were seen to move only within a single rate component. Moreover, proteins that have common functions or interact with each other tend to be moved together. These observations led to a new view of axonal transport, the structural hypothesis [7]. This model can be stated simply proteins and other molecules move down the axon as component parts of discrete subcellular structures rather than as individual molecules (Table 28-1). 

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... 

Separation of a mixture of proteins by electrophoretic techniques such as polyacrylamide gel, SDS polyacrylamide or iso-electric focusing usually results in a complex pattern of protein bands or zones. Interpretation of the results often involves a comparison of the patterns of test and reference mixtures and identification of an individual protein, even using immunoelectrophoresis (Figure 11.15), is very difficult. However, specific proteins can often be identified using an immunoblotting technique known as Western blotting. The prerequisite is the availability of an antibody, either polyclonal or monoclonal, against the test protein. 

Urea is added to each incubation to separate the two strands of DNA and the single strands are separated electrophoretically in adjacent lanes of an SDS polyacrylamide gel. Urea is also present in the gel to ensure that the strands of DNA stay separated. The gel is then autoradiographed. The DNA sequence can be deduced from the ascending order of the bands in the four adjacent lanes. 

A partially purified HIV viral lysate is laid onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel slab and then electrophoresed, which distributes the HIV peptides through the gel by their relative molecular mass. The higher-molecular-mass proteins form bands near the top of the gel. The proteins on the gel are then transferred electrophoretically onto nitrocellulose paper. The paper is sliced into thin strips, each having the full distribution of HIV antigen bands. The strip is used as a solid support of an indirect immunoassay, and antigen-antibody reactions form insoluble colored bands on the strip. 

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