SDS-polyacrylamide-gel electrophoresis

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)...

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. 

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... 

Figure 4. SDS-polyacrylamide gel electrophoresis of microsomes from variously pretreated rainbow trout (A), control microsomes, 90 fig protein/gel (B), /3-naphthoflavone-inauced microsomes, 90 fig protein/gel (C), Aroclor 1242-induced microsomes, 90 fig protein/gel.

The component subunits of PA700 range in size from 112 to 28 kDa. The S nomenclature identifies subunits on the basis of their relative mobility during SDS polyacrylamide-gel electrophoresis, whereas the Rpt/Rpn nomenclature distinguishes between the AAA ATPase subunits (Regulatory particle triple-A protein) and the non-AAA ATPase subunits (Regulatory particle non-ATPase) (see below). 

Figure 3.12 Phosphate transfer between the protein histidine kinase HupT and the response regulator HupR. HupT (7 pmol) was phosphorylated at 30°C with [y- PJATP for 5 min, before addition of wild-type (HupR) or mutated (HupR-DS4E) HupR protein. Aliquots were withdrawn 5 min or 20 min after addition of HupR and analysed by SDS polyacrylamide gel electrophoresis. HupT20 HupT was phosphorylated for 20 min in the absence of HupR.

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... 

Immune complexes are solubilised in 50 xl of 2X Laem mli buffer, boiled and subjected to SDS-polyacrylamide gel electrophoresis or washed once with the enzyme assay buffer and used for enzymatic assays. 

The flanking fractions of the main IgG peak may contain small quantities of contaminating proteins. Each fraction should be analyzed by SDS-polyacrylamide gel electrophoresis before the desired fractions are pooled (see Chapter 2, Note 15). 

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). 

Figure 2. SDS-polyacrylamide gel electrophoresis of the purified CBH I and CBH I core fragment using the Pharmacia PhastSystem.

Hl-trap Benzamidine EE column SDS-polyacrylamIde gel electrophoresis system Thrombin BOO units In 0.5 ml PBS (stored at -80°C) Eactor Xa 400 units In water to give a final solution of 1 Unlt/pil stored at-80°C... 

Sterile microcentrifuge tubes Sterile pipettes and pipette tips SDS-polyacrylamIde gel electrophoresis system... 

---