Thin layer gel filtration

Delincee and Radola100 used a commercial preparation, as well as fresh tomatoes, for the preparation, purification, and characterization of tomato pectinesterase. The tomatoes were pressed and then homogenized directly with ammonium sulfate at 70% saturation. The precipitate obtained was extracted with 0.3 M phosphate and repeatedly salted out with ammonium sulfate, and the product was separated on a column of Sephadex G-75. The pattern of separation was similar to that in preceding work.50,97 A detailed study of the size properties of pectinesterase was conducted by gel-filtration and sedimentation analysis.100 By column and thin-layer gel-filtration on Sephadex G-75, the approximate molecular weight of a number of preparations of tomato pectinesterase was determined, values of 24,000 and 27,000 being obtained. A possible interaction of the... 

Molecular weight (by thin-layer gel-filtration chromatography)... 

A8. Ahlstedt, S., Hanson, L. A., and Wadsworth, C., A Clq immunosorbent assay compared with thin layer gel filtration for measuring IgG aggregates. Scand. ]. Immunol. 5, 293-298 (1976). 

Gel filtration and sedimentation generally have less resolving power and sensitivity than gel electrophoresis but they can be more easily scaled up for moderate and large scale preparative use. Thin layer gel filtration can be moderately sensitive for analytical applications and analytical ultracentrifugation can provide the fastest analysis as well as being reasonably sensitive. 

JIO. Johnson, B. G., and Rymo, L., Separation of proteins by thin layer gel filtration. Acta Chem. Scand. 18, 217-223 (1964). 

Thin layer chromatography may be either carried out by the adsorption principle (if the thin layer is prepared by an adsorbent such as Kieselguhr, or alumina) or by the partition principle (if the layer is prepared by a substance such is silica gel which holds water like the paper). More recently, thin layer gel filtration chromatography Is also performed as we will see later. In this case, the layer is made up of controlled pore beads of various gels. 

Thin layer gel chromatography. Determann, and Johanson and I nno (in 1962) showed that gel permeation chromatography could be conducted using thin layers of gel. For clinical use, thin layer gel filtration (TLG) is ideal since very small sample volinne is required for this technique. In this technique, a layer of hydrated gel is applied to the plate. There is no need of a fixative to adhere the gel beads. The plate is not dried at all and is placed in an airtight container at an angle of 20 . The plate is connected to reservoirs at either end by means of filter paper bridges. Equilibrium must be carried out for at least 12 hours. Such equilibrium serves to normalize the ratio between the stationary and mobile phase volumes. The sample may be applied either as a spot or as a band. TTie plate may then be developed for a suitable time and the sep2u-ated components detected by suitable means. 

Hung, C. H., Strickland, D. K., and Hudson, B. G. (1977). Estimation of molecular weights of peptides and glycopeptides by thin layer gel filtration. Anal. Biochem. 80 91-100. 

Radioiodide has also been separated from iodine-labeled proteins (human serum albumin and Bovine IgG) by thin-layer gel filtration on Sephadex G-200 and G-75 superfine layers (163). (Sephadex is a modified dextran.) The protein solutions were applied in amounts of 5-10 vJL to the Sephadex layers and the angle of slope of the layers was 15°. Elution was carried out with 0.2 M Tris buffer (Tris = tris (hydroxymethyl) aminomethane) at pH 8.0. The buffer contained Tween 80. Separations took 45-60 min on Sephadex G-75 and 3-4 h on Sephadex G-200 and were particularly good on the latter. 

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