Purification gel filtration

L. Hagel, "Gel Filtration" in J-C. Janson and L. Ryden, ed.. Protein Purification Principles, High Resolution Methods, and Applications, VCH Pubhshers,... 

Purification of poloxamers has been extensively investigated due to their use in medical applications, the intention often being to remove potentially toxic components. Supercritical fluid fractionation and liquid fractionation have been used successfully to remove low-molecular weight impurities and antioxidants from poloxamers. Gel filtration, high-performance liquid chromatography (HPLC), and ultrafiltration through membranes are among the other techniques examined [5]. 

Step 1. Gel filtration on Sephadex G-100, in 10 mM sodium phosphate buffer, pH 6.5, containing 5mM EGTA and 0.2 M NaCl, which was the basic buffer used throughout the purification process. 

Water soluble protein with a relative molecular mass of ca. 32600, which particularly contains copper and zinc bound like chelate (ca. 4 gram atoms) and has superoxide-dismutase-activity. It is isolated from bovine liver or from hemolyzed, plasma free erythrocytes obtained from bovine blood. Purification by manyfold fractionated precipitation and solvolyse methods and definitive separation of the residual foreign proteins by denaturizing heating of the orgotein concentrate in buffer solution to ca. 65-70 C and gel filtration and/or dialysis. 

Extraction of Sodium Channel Blockers. A review of published reports shows that methods for purification of sodium channel blockers from bacterial cultures are similar to techniques for isolation of TTX and STX from pufferfish and dinoflagellates (30, 31, 38, 39). Typically, cell pellets of bacterial cultures are extracted with hot 0.1% acetic acid, the resulting supernatant ultra-filtered, lyo-philized, and reconstituted in a minimal volume of 0.1% acetic acid. Culture media can also be extracted for TTX by a similar procedure (Ji). Both cell and supernatant extracts are analyzed further by gel filtration chromatography and other biological, chemical, and immunological methods. Few reports describe purification schemes that include extraction of control samples of bacteriological media (e.g., broths and agars) which may be derived from marine plant and animal tissues. 

Acetyl esterase (AE) has been purified to homogeneity from orange peels. The purification steps included cation exchange chromatography and gel filtration. The enzyme has affinity for triacetin and sugar beet pectin with K, of 39 mM and of 26 mg/ml, respectively. AE has a MW of 42 kD and is a monomer. The isoelectric point is at pH > 9. 

In order to remove the residual PME the AE fraction was further fractionated on Mono S cation exchange column. Unlike the DEAE-Sepharose column, where PME elutes after the AE activity, the order of elution was reversed on the Mono S column (Fig. 1). The last step in the purification was a gel filtration (Sephacryl S-200) column. 

Miller and Macmillan [4] carried out purification of pectinesterase from Fusarium oxysporum f. sp. vasinfectum culture fluid (fivefold degree of purification). According to the obtained data the purified enzyme possessed very low polygalacturonatlyase one. Disk electrophoresis at pH 4.3 revealed two protein components. The authors did not study distribution of pectinesterase activity in these components. Molecular weight of fungal pectinesterase determined using gel — filtration on Sefadex G — 75 was found to be 35,000. 

After the release of the oligosaccharides, they must be purified by a variety of methods before structural analyses can be undertaken. Ion-ex-change chromatography, gel-filtration chromatography, or some type of electrophoresis is usually used for the purification. At this point, structural analyses may begin. 

Purify the thiolated protein from unreacted Traut s reagent by gel filtration using your buffer of choice (i.e., 20mM sodium phosphate, 0.15M NaCl, ImM EDTA, pH 7.2). The addition of EDTA to this buffer helps to prevent oxidation of the sulfhydryl groups and the resultant disulfide formation. After purification, use the thiolated protein immediately... 

Purify the sulfhydryl-modified protein by dialysis against 50mM sodium phosphate, 1 mM EDTA, pH 7.5, or by gel filtration on a Sephadex G-25 column using the same buffer. Again, if a peptide of low molecular weight is being modified, use careful gel filtration for purification. 

Purify the modified protein from reaction by-products by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. Alternatively, centrifugal spin columns containing a desalting resin may be used for rapid purification (Thermo Fisher). 

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