Salt gradient

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

W. L., and Rivera, K. E., Facile purification of fibrinogen fragments using a computer-based model with general applicability to the generation of salt gradients, Prot. Expression Purif., 14, 71, 1998. 

After sample loading, the cation-exchange RAM-column was placed in-line with the analytical cation-exchange column and analytes were eluted with a salt gradient. A total of 24 fractions of 4 min duration (2 mL of eluent) were transferred to the... 

Physical separation methods can be based on equilibrium considerations, but the majority are not. Ordinary filtration is an example of a non-equilibrium, physical method and so is ordinary centrifugation— e.g.—the separation of a precipitate from the suspending liquid using an artificial gravity field. There are separation methods, which are called filtration which are not such as gel filtration. Ultracentrifugation in a salt gradient is a physical equilibrium method. 

Figure 5.1 Comparison of IgM complexed with DNA versus IgM decomplexed by pretreatment with 1.0 M NaCl and 0.05 M EDTA. Sample purified IgM plus partially degraded DNA extracted from the host cell line. Dotted trace marks the salt gradient. Arrow marks introduction of 5 mM EDTA to the salt strip. Mono-S HR5/5, pH 5.5. See text for explanation. (Data from P. Gagnon, 1996, Special weapons and tactics for removal of product-bound DNA, oral presentation, BioEast 96, Washington, D.C.)...

It is impossible to recommend how far you should pursue the possibilities discussed in this chapter. Essentially, all analytical process developers routinely screen both anion and cation exchangers with salt gradients over a range of fixed pH values. A smaller subset routinely evaluates pH gradients as well. Exploration into more exotic territory is usually undertaken only when conventional approaches fail. This is probably as it should be. As powerful and versatile as IEC is, it is only one of a suite of proven chromatography techniques for protein analysis. If investigating the basics in IEC does not yield the results you seek, it... 

This salt-gradient theory deserves further attention. It is possible that the concepts may be applicable to vapor explosions in different industries. 

One experiment which does not seem to fit into the network of the salt-gradient theory was that of Wright and Humberstone (1966), who impacted water on molten aluminum and obtained explosions. These results are at variance with those of Anderson and Armstrong, but the latter worked at 1 bar whereas the former used a vacuum environment. It might be possible that, under vacuum, it is much easier to achieve intimate contact between the aluminum and water and, under these conditions, there may be sufficient reaction between the aluminum and water to allow soluble aluminum salts to form. This salt layer could then form the superheated liquid which is heated to the homogeneous nucleation temperature and explodes. 

Rotor/run conditions SW 55 Ti rotor at 55000 rpm for approximately 6 hours. These recommendations form the core of any procedure. SpinPro usually considers more factors in the rotor selection process than does the expert. In determining the run speed, SpinPro considers every possible reason to reduce the run speed. If there are none, the rotor is run at full speed. When there are reasons (e.g., when using salt gradients, bottles, differential pelleting, or discontinuous runs), the run speed may have to be reduced dramatically, from 80,000 rpm to 40,000 rpm, for example. There are many cases of rotors being run too slow for the application or too fast for safety. Accurate determination of the run time is a complex problem based on the gradient characteristics, calculations, interpolations from numerical tables, and experience. SpinPro employs all of these methods in order to infer run times for many special cases. 

The CALCULATION function provides a variety of routine calculations performed in most ultracentrifugation laboratories. Included are dilution calculations for sucrose, a pelleting time calculation, and a calculation for determining rotor speed reductions for salt gradients. As with the INFORMATION function, the CALCULATION function is a support tool in the effort to efficiently design and carry out a separation. 

Initial Fractionation. The most effective method found for the initial fractionation of the complex enzyme mixtures present in wood-culture extracts was anion exchange chromato phy eluting with salt gradients (9,74,75). This permitted further sample concentration by functioning as a solid phase extractant during loading. It also showed higher sample capacity and better separations than other methods. 

Figure 5. Optical absorbance at 280nm and 409nm (denoting heme-iron containing ligninases) for fractions from the preparative DEAE anion exchange column and salt gradient used to fractionate the enzymes in a crude culture extract. (Reproduced widi permission from ref. 15. Copyright 1990 Springer-Verlag.)...

Figure 4. Elution profile of the 2 - and 5 -0-glucosyltransferases after chromatography on Brown 10X agarose column using pH-salt gradient. Insert autoradiographed enzyme reaction products.

Two-dimensional lECxRP setups by using an ion exchange column by means of salt gradient in the first dimension and an RP column in the second dimension have been extensively employed for... 

Simultaneous optimizatiou of the steepuess of a salt gradient, column length, and mobile phase flow rate using iso-resolution plots was recently applied to cation-exchange chromatography of proteins on short columns [129,130]. 

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