Fractionation gel filtration

Thirty-two years later, Ko jima etal. (2000a) purified Latia luciferase by the following steps ammonium sulfate fractionation, gel-filtration, affinity chromatography and Mono-Q anion-exchange FPLC. 

If a further purification is desired, then the supernatant (above) can be subjected to ammonium sulfate fractionation, gel filtration on Sephadex G-200, and finally on a-aminopropane-agarose affinity column. On polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, the final product migrated as a single band with an estimated molecular weight of 113,000. Upon sedimentation equilibrium velocity ultracentrifugation, an estimated molecular weight value of near 117,000 was obtained. 

In this laboratory, attempts (G6, G8) have been made to purify and crystallize human placental alkaline phosphatase enzyme by a number of procedures involving homogenization with 0.05 M Tris buffer (pH 8.6), extraction with butanol, ammonium sulfate precipitation, exposure to heat, ammonium sulfate fractionation, dialysis, repeated ethanol fractionation, gel filtration with Sephadex G-200 (Fig. 18), continuous curtain electrophoresis on paper (Beckman Model CP), multiple TEAE-cellulose anion exchange chromatography, and equilibrium dialysis. Variant A (electrophoretically fast-moving) of human placental alkaline... 

An endo- -> 3)-jS-D-glucanase (EC 3.2.1.6), isolated from the culture filtrate of the fungus Mucor hiemalis, was purified by ammonium sulphate fractionation, gel filtration, and ion-exchange chromatography. The optimum pH, optimum temperature, and Km value of the enzyme were 5.0, 50 C, and 0.048%, respectively. The enzyme (mol. wt. 3.0 x 10 by gel filtration) was strongly inactivated by Pb +, Cu +, and Hg + ions and also inhibited by Zn + and Fe + ions. The enzyme was specific for laminaran and the action pattern of the enzyme was of the endo-type. 

Myeloma proteins are ordinarily isolated by salt fractionation, gel filtration, and either ion-exchange chromatography or zone electrophoresis. H and L chains ire obtained from the purified proteins by reduction of interchain disulfide bonds, followed by gel filtration in a solvent which disrupts noncovalent interactions between the chains. Bence Jones proteins are another major source of L chains. 

With this simple method of subcellular fractionation, gel filtration and prep disc acrylamide gel electrophoresis, it is possible to obtain highly purified HPL from fresh placentas. 

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