Separation by Electrophoresis

Electrophoresis separates charged proteins on the basis of their different mobilities in an electric field. When a solution of proteins is subjected to an electrical potential, the 

CHAPTER 3 Protein Isolation and Determination of Amino Acid Sequence 

A schematic diagram of the steps in the purification of a protein fused with glutathione S-transferase. Protein purification using this procedure utilizes three columns and is fully automatic. 

Electrophoretic techniques also yield estimates of the molecular weights of proteins and nucleic acids. The detergent sodium dodecyl sulfate (SDS) and proteins form 

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Once the fragments have been separated by electrophoresis (step... 

Ultimately, however, it should be noted that these examples of classical gel electrophoretic separations are batch processes and therefore limited in sample throughput. To achieve true preparative-scale separations by electrophoresis, it becomes necessary to convert to continuous processes. 

Figure 7-11. Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in human serum. LDH isozymes of serum were separated by electrophoresis and visualized using the coupled reaction scheme shown on the left. (NBT, nitroblue tetrazolium PMS, phenazine methylsulfate). At right is shown the stained electropherogram. Pattern A is serum from a patient with a myocardial infarct B is normal serum and C is serum from a patient with liver disease. Arabic numerals denote specific LDH isozymes.

The isoenzymes can be separated by electrophoresis on cellulose acetate, and Roberts, et al ( ) have described a method whereby the separated isoenzymes are eluted and then assayed kinetically. 

CK isoenzymes have been separated by electrophoresis on cellulose acetate, agar gel, agarose and polyacrylamide gel. 

Various methods have been used to examine the composition of proteins adsorbed to SAMs. Overall adsorption patterns can be examined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [50, 76, 77]. Absorbed proteins are eluted from the surface with surfactant (SDS), and then separated by electrophoresis. The proteins of interest are examined by western blotting [50, 76, 77]. Protein-specific antibodies can be used to detect proteins of... 

What general characteristic must a component have to be separated by electrophoresis ... 

Another similar technique to Southern blotting is Northern blotting. Here, instead of DNA fragments, mRNA fragments are probed with a labelled cDNA probe after separation by electrophoresis and transfer to nitrocellulose membranes. Northern blotting is used to detect and quantify mRNA from tissue extracts. 

Fluorescein-labelled deoxyribonucleotide triphosphates may be used in place of those labelled with 32P. Once the DNA sequences are separated by electrophoresis, the resulting DNA bands fluoresce and are analysed by a flu-orogram imager, which produces a picture of the fluorescent bands similar to the autoradiography produced when using 32P-labelled nucleotides. 

VLDL) comprising apolipoproteins B and E, combined mainly with triglycerides are secreted from the liver. Apo A is an a globulin and apo B is found in the (3 fraction, thus HDL and LDL are sometimes referred to as a and (3 lipoprotein respectively and VLDL because in separation by electrophoresis at pH 8.6, it runs ahead of LDL is called pre- 3 lipoprotein. 

Separation by electrophoresis is based on differences in solute velocity in an electric field. The velocity of an ion is given by... 

The resultant different complexes with the same stoichiometry [SL(1) and SL(2)] generally can t be separated by electrophoresis because of the same size and charge. Only the macroscopic equilibrium constants and K2 can be derived from electrophoresis data. 

Fig. 24. The yield of UpU and UpC as functions of dose at 280 nm. The photo-products were separated by electrophoresis 24 hr after the end of the irradiation. Yields are given as a percentage of CpC (Freeman, Hariharan, and Johns71).

Haug75 has reported that the concentration of TpC reaches a photostationary state at 280 nm and pH 7 at about half its initial value. Three photoproducts, separable by electrophoresis and stable to heat, are formed. The quantum yield for decay of TpC is independent of wavelength of light, and is 0.006 at pH 7 and 0.002 at pH 2. Heating the solution at 80°C restores about 40% of the absorption loss at 270 nm for the solution irradiated at pH 2 and about 70% of the loss for solutions irradiated at pH 7. Irradiation at 240 nm does not increase the absorption at 270 nm. One would expect dimers and hydrates to be formed in the photolysis, but the observations summarized here are difficult to reconcile with such products exclusively and suggest deep-seated irreversible changes. 

Before discussing the various derivatives that have been used, it should be remembered that oligosaccharides are often obtained by chromatography on paper or on cellulose columns, and this may cause them to become contaminated by xylan this may be eliminated by extraction of the crude fractions with hot ethanol.474 For similar reasons, glucose that is really extraneous may be detected in fractions separated by electrophoresis on cellulose acetate.475 Gas-liquid chromatography was used to show the presence in pituitary glyco-... 

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

GAGs are separated by electrophoresis to yield a distinct pattern for the different MPS types [32, 33]. It was found that electrophoresis in barium acetate depends largely on the structure of the GAGs, whereas the degree of sulfatation is of minor importance [69]. One-dimensional electrophoresis, as described here, offers lower resolution than two-dimensional techniques [28], but is simpler to evaluate and allows the assessment of several patients in one run. 

In the first dimension, 20 [tl plasma is separated by electrophoresis at 4°C in a 0.75% agarose gel using a 1 2 16 dilution of a barbital buffer. Bromophenol blue is added to a standard sample to visualize albumin in the native gel. The electrophoresis is stopped when the albumin/bromophenol blue marker has migrated 6 cm. Agarose gel strips containing the preseparated lipoproteins are then transferred to a 4-20% polyacrylamide gradient gel. Separation in the second dimension is performed at 40 mA for... 

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