Sephadex

Abstract: 1. Correlation between elution volume, V(e), and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (alpha-crystallin) on Sephadex G-200 columns at pH7.5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V(e)-log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V(e)-log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of ;typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including gamma-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10(6). 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of gamma-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.

Abstract: A method is described for measuring glomerular permeability by dextran clearance using Sephadex gel filtration technique. Clearance is shown to be a function of molecular size, decreasing with increasing molecular weight. At a mol. weight higher than 55,000, the clearance is very low and approximately zero, while it averages 95 ml per minute at a mol. weight of 15,000. If plotted in a log.log. graph, a linear relation is found between clearance and molecular weight. In a semilog. graph, however, with clearance plotted against log. Stokes radius, the relation is also linear, with a slight deflection in the high molecular part, however.

Abstract: We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.