Electrophoresis preparative

i % hydroxyethyl cellulose. Run 600 V (current 58 pA). The three major peptides (pi 6.1. 6.93, and 3.95) were eluted and analyzed by mass spectrometry 175]. 

Fignre 12. Preparative lEF in granulated Sephadex layers A) Small-scale separation B) Large-scale separation a) Electrode b) Filter paper pad soaked in electrode solution c) Cooling block d) Glas.s plate e) Gel layer f) Focused proteins g) Trough 1178). 

The arrows indicate the fluid flow pattern. At the end of the process, the flow pattern is switched to the harvesting position toward the fraction collector 183] a) Focusing cell b) Fraction collector c) Bubble trap d) Peristaltic pump e) Heat exchanger f) 30-Channel manifold 

In the case shown, a protein with pi 6..S is trapped between the pi 6 anodic and pi 7 cathodic membranes, whereas proteins with higher pi move toward the cathode and a species with lower pi (5.5) crosses the two membranes and collects at the anode [128]. 

190) M J Dunn (ed ) 2-D PAGE 91. Zebra Printing. Peri-vale 1991, pp. I-.325. 

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CE is generally more suited to analytical separations than to preparative-scale separations. However, given the success of CE methods for chiral separations, it seems reasonable to explore the utility of preparative electrophoretic methods to chiral separations. Thus, the purpose of this work is to highlight some of the developments in the application of preparative electrophoresis to chiral separations. Both batch and continuous processes will be examined. 

Porath, J, Some Recent Developments in Preparative Electrophoresis and Gel Filtration, Metabolism 13, 1004, 1964. 

Figure 4. Purification of PemB from E. coli K38 pGPl-2/pPME6-5 cells. Proteins were separated by urea-SDS-PAGE. Lane 1, induced cell lysate lane 2, soluble protein fraction from induced cells lane 3, membrane fraction from non-induced cells lane 4, membrane fraction from induced cells lane 5, membrane proteins not extracted by Triton X-100 lane 6, membrane proteins extracted by Triton X-100 lane 7, PemB purified by preparative electrophoresis. The molecular weight standard positions are indicated.

R. M. C. Sutton and A. M. Stalcup. Preparative Electrophoresis. Encyclopedia of Separation Science, Academic Press Ltd (1999) in press. 

The N-linked pentasaccharide core Man3(GlcNAc)2 glycopeptide bearing the extracellular MMP inducer sequence 517 (emmprin 34—58) has been successfully linked to G(l) PAMAM aminodendrimer by thioester activation (Fig. 63). The resulting octameric 30 kDa construct was obtained in low yield, but was purified by preparative electrophoresis and fully characterized by MALDI-TOF mass spectrometry. The multivalent architecture was built to evaluate the requirement of emmprin multimerization for inducing MMP expression.384... 

Preparative electrophoresis on Sephadex G-25 (Ref. 168) or double isoelectric focusing,208 preceded by chromatography on Sephadex G-75, CM-cellulose, and calcium phosphate, was used for the isolation of endo-D-galacturonanase from the filtrate of a Verticillium albo-atrum culture. The homogeneity was confirmed in both cases by electrophoresis on poly(acrylamide) gel. The molecular weight of the enzyme was close to the values found for Aspergillus endo-D-galacturonanases. 

Jayle, Herman-Boussier, and Moretti have shown that different types of human Hp in amounts large enough for analysis can be prepared by fractional ammonium sulfate precipitation combined with a rather conventional, preparative electrophoresis in an acetate buffer of pH 5.8. Schultze and Heide (S2) utilize a more complicated procedure, in which preparative electrophoresis (acetate buffer, pH 4.4) is likewise the final step. 

Perez-Ruiz, T., Martinez-Lozano, C., and Galera, R. (2006). Development and validation of a capillary electrophoresis method with laser-induced fluorescence detection for the determination of captopril in human urine and pharmaceutical preparations. Electrophoresis 27(12), 2310—2316. 

Triton X-100, EDTA, dithiothreitol and electrolyte protect enzyme in dilute solution and against denaturation by heat or extreme pH-values [12, 48] <2>, at low dithioerythritol concentrations enzyme tends to aggregate [5] <2>, bovine serum albumin, 1 mg/ml, stabilizes dilute enzyme solutions [5] <2>, diadenosine pentaphosphate, i.e. AP5A, stabilizes during preparative electrophoresis [7]... 

Following digestion, purify the D NA by preparative electrophoresis on a 1% agarose gel. The cut vector band of 4.7 kb is excised, purified from the gel, precipitated, and quantitated... 

Another recent technique, preparative electrophoresis, is recycling isoelectric focusing developed by Bier and colleagues (University of Arizona). See Fig. 2. 

Fully equipped protein purification laboratories should also have preparative electrophoresis and isoelectric focusing apparatuses for rare occasions when other techniques fail to give sufficient separation. 

Figure 9. Results of simultaneous preparative electrophoresis of equal samples (0.5 ml.) of plasma obtained from blood taken 0, 2, and 5 hours after start of liver perfusion (Figure 8)...

Masuoka, J., Glee, P.M., and Hazen, K.C. (1998). Preparative isoelectric focusing and preparative electrophoresis of hydrohobic Candida albicans cell wall proteins with in-line transfer to polyvinylidene difluoride membranes for sequencing. Electrophoresis 19, 675-678. 

It should also be mentioned that glycopeptides closely related to the pep-tido-polysaccharide of human-strain, wax D fractions are found in the culture filtrate of tubercle bacilli and in aqueous extracts thus, Kdra and Keil have described a glycopeptide, isolated from culture filtrates of virulent, human strains of M. tubercidosia, which could be purified by preparative electrophoresis and which contains alanine, a, -diaminopimelic acid, and glutamic acid in equimolecular proportions structure (22) was proposed for the peptide portion of this glycopeptide. 

Total tRNA and 2% tRNA from yeast and bull s liver were obtained according to a previously described procedme [5]. An enriched fraction of specific tRNA was obtained by preparative electrophoresis in polyacrylamide gel [6]. The enriched tRNA preparations contained from 75 to 150 nmoles of tRNA. 

Few issues of the analytical journals go by without the appearance of a new design for gel electrophoresis apparatus, and there are no doubt many satisfactory alternatives to the three versions that we have discussed. Microelectrophoresis and preparative electrophoresis pose special problems, and will be considered separately ( 7.S.2.2 7.S.2.3 ch. 9). 

The first problem in all methods of preparative electrophoresis is the location of the zones. A sure but tedious method is to slice the gel... 

Fig. 9,2. Small-scale preparative electrophoresis device. Buffer is swept through the gap between the upper (A) and lower (B) parts of the cylindrical gel, and then to a fraction collector (Mann and Huang 1969),...

Fig. 9.3. Small-scale preparative electrophoresis apparatus by Popescu et al. (1971), The glass tube containing the gel (0.8 cm o.d.) fits into the sawn-off barrel of a 2 ml disposable plastic syringe, in which have been cut 2 mm wide slots. A peristaltic pump, working on the outlet tube causes buffer to be drawn through the compartment made up by the syringe bottom.

Fig. 9.4. Densitometer traces of stained poly(rA) zones after electrophoresis in 2.4% gels, a) Typical unfractionated sample, b) Fractions prepared by salt precipitation, c) Fractions made by preparative electrophoresis. The broken line is the profile of tRNA. Intensities are normalised at the peak. Mobilities are relative to bromophenol blue. The arrow indicates the end of the gel (from Finder and Gratzer 1974).

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