Separator Protein

Fig. 8. A simplified combinatorial approach to identify the binding sequence for a (O) protein of interest, within 4096 sequences where ( ) represents the PCR primers, N a nucleotide, ie. A, G, C, or T. An essential aspect of this experiment is the abiHty to separate protein-bound from free DNA prior to...

Ultrafiltration. Membranes are used that are capable of selectively passing large molecules (>500 daltons). Pressures of 0.1—1.4 MPa (<200 psi) are exerted over the solution to overcome the osmotic pressure, while providing an adequate dow through the membrane for use. Ultrafiltration (qv) has been particulady successhil for the separation of whey from cheese. It separates protein from lactose and mineral salts, protein being the concentrate. Ultrafiltration is also used to obtain a protein-rich concentrate of skimmed milk from which cheese is made. The whey protein obtained by ultrafiltration is 50—80% protein which can be spray dried. 

The size separation of proteins has been routinely called gel filtration because of the historic use of cross-linked gels for this application. Specially modified Zorbax PSM columns, Zorbax GF-250 and Zorbax GE-450, are used for separating proteins by size. These columns are packed with porous silica micro-... 

As with other size-exclusion techniques, the pore size of the selected Zorbax GF column should provide resolution over the molecular size range of the proteins that are to be separated. The Zorbax GF-250 column separates proteins in the range of 4000 to 400,000 Da. The Zorbax GF-450 provides separation over the range of 10,000 to 1,000,000 Da. When these two columns are coupled, they can be used to separate proteins with molecular weights of 4000 to 1,000,000. 

Sucrose gradients for separation of membrane proteins must be able to separate proteins and protein-lipid complexes having a wide range of densities, typically 1.00 to 1.35 g/mL. 

Application of charges and filtration may separate protein very efficiently. Electro-kinetic deposition uses voltage gradients of 1050V/cm to produce solid biomass with densities of up to 40% w/v. 

Specificity of the antisera was assessed by Western blotting. Electrophoretically separated proteins from culture filtrates were transferred to 0.45 fim nitrocellulose membranes. After transfer of proteins, membranes were... 

Kamel, H., Brown, D.H., Ottaway, J.M. and Smith, W.E. (1977) Determination of gold in separate protein fractions of blood serum by carbon furnace atomic-absorption spectrometry. Analyst, 102, 645-663. 

Under conditions of copper deficiency, some methanotrophs can express a cytosolic, soluble form of MMO (sMMO) (20-23), the properties of which form the focus of the present review. The sMMO system comprises three separate protein components which have all been purified to homogeneity (24,25). The hydroxylase component, a 251 kD protein, contains two copies each of three subunits in an a 82y2 configuration. The a subunit of the hydroxylase houses the dinuclear iron center (26) responsible for dioxygen activation and for substrate hydroxylation (27). The 38.6 kD reductase contains flavin adenine dinucleotide (FAD) and Fe2S2 cofactors (28), which enable it to relay electrons from reduced nicotinamide adenine dinucleotide (NADH) to the diiron center in the... 

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. 

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.

These columns have been used for separation of proteins of over 200 kDa MW in our experiments as shown by analysis using a ID gel. In addition, columns with larger particle sizes have been used to separate proteins of over 400 kDa (55-56). The NPS RP-HPLC method provides a liquid phase method for separating large intact proteins for further analysis. More specifically, it provides a means of separating proteins for interfacing to mass spectrometric analysis. 

Complex viruses Some virions are even more complex, being composed of several separate parts, with separate shapes and symmetries. The most complicated viruses in terms of structure are some of the bacterial viruses, which possess not only icosahedral heads but helical tails. In some bacterial viruses, such as the T4 virus of Escherichia coli, the tail itself is a complex structure. For instance, T4 has almost 20 separate proteins in the tail, and the T4 head has several more proteins. In such complex viruses, assembly is also complex. For instance, in T4 the complete tail is formed as a subassembly, and then the tail is added to the DNA-containing head. Finally, tail fibers formed from another protein are added to make the mature, infectious virus particle. 

Seow TK et al. Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated proteins by... 

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