Chromatography sepharose

Column Chromatography. Sepharose beads containing covalently linked gangliosides (0.2 ml packed volume) were placed into a pasteur pipette containing a small amount of glass wool. Columns were washed with HEM containing 50 ug/ml bovine serum albumin (3 ml). Interferon solutions in MEM-albumin (1 ml) were placed on the columns, which were eluted with MEM-albumin at a flow rate of no more than one drop per minute. Fractions of 1 ml were collected and interferon titers determined in each fraction after serial two-fold dilution. Columns onto which mouse fibroblast interferon had been loaded, were eluted with MEM-albumin first, then with 0.07 M N-acetylneuraminyl lactose at pH 2. 

A wide variety of immobilized antigens, chemicals, and receptor molecules have been used effectively for affinity cell chromatography. Sepharose beads coated... 

Sepharose (e.g. Sepharose CL and Bio-Gel A) is a bead form of agarose gel which is useful for the fractionation of high molecular weight substances, for molecular weight determinations of large molecules (molecular weight > 5000), and for the immobilisation of enzymes, antibodies, hormones and receptors usually for affinity chromatography applications. 

Angiotensinogen (from human blood serum) [643I5-I6-8]. Purified by chromatography on Blue Sepharose, Phenyl-Sepharose, hydroxylapatite and immobilised 5-hydroxytryptamine [Campbell et al. Biochem J 243 121 1987]. 

Avidin (from egg white) [1405-69-2] Mr -70,000. Purified by chromatography of an ammonium acetate soln on CM-cellulose [Green Biochem J 101 774 1966]. Also purified by affinity chromatography on 2-iminobiotin-6-aminohexyl-Sepharose 4B [Orr 7 Bio/C/iew 256 761 1981]. It is a biotin binding protein. 

Cathepsin B (from human liver) [9047-22-7] Mr 27,500 [EC 3.4.22.1]. Purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal-linked to Sepharose 4B, with elution by 2,2 -dipyridyl disulfide [Rich el al. Biochem J 235 731 1986 Methods Enzymol 80 551 1981]. 

EC 1.15.1.1]. Purified by DEAE-Sepharose and copper chelate affinity chromatography. The preparation was homogeneous by SDS-PAGE, analytical gel filtration chromatography and by isoelectric focusing [Weselake et al. Anal Biochem 155 193 1986 Fridovich J Biol Chem 244 6049 7969]. 

Dipeptidyl aminopeptidase (from rat brain) [9031-94-1] [EC 3.4.11.10]. Purified about 2000-fold by column chromatography on CM-cellulose, hydroxylapatite and Gly-Pro AH-Sepharose. [Imai et al. J Biochem (Tokyo)93 431 1983.]... 

Hydroxy butyrate dehydrogenase (from Rhodopseudomonas spheroides) [9028-38-0] Mf 85,000, [EC 1.1.1.30], amorphous. Purified by two sequential chromatography steps on two triazine dye-Sepharose matrices. [Scavan et al. Biochem J 203 699 7952.]... 

EC 1.1.1.27]. 40-Fold purification by affinity chromatography using Sepharose 4B coupled to 8-(6-aminohexyl)amino-5 -AMP or -NAD. [Lees et al. Arch Biochem Biophys 163 561 7974 Pesce et al. J Biol Chem 239 1753 7964.]... 

Lipoprotein lipase (from bovine skimmed milk) [9004-02-8] [EC 3.1.1.34]. Purified by affinity chromatography on heparin-Sepharose [Shirai et al. Biochim Biophys Acta 665 504 1981]. 

Lipoteichoic acids (from gram-positive bacteria) [56411-57-5J. Extracted by hot phenol/water from disrupted cells. Nucleic acids that were also extracted were removed by treatment with nucleases. Nucleic resistant acids, proteins, polysaccharides and teichoic acids were separated from lipoteichoic acids by anion-exchange chromatography on DEAE-Sephacel or by hydrophobic interaction on octyl-Sepharose [Fischer et al. Ear J Biochem 133 523 1983]. 

Purified by dissolving in Triton X-100 and deoxycholate, and by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose [Grondal and Zimmerman Biochem J 245 805 1987]. 

Protamine kinase (from rainbow trout testes) [37278-10-7] [EC 2.7.1.70]. Partial purification by hydoxylapatite chromatography followed by biospecific chromatography on nucleotide coupled Sepharose 4B (the nucleotide was 8-(6-aminohexyl)amine coupled cyclic-AMP). [Jergil et al. Biochem J139 441 1974.]... 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

Purified by (NH4)2S04 fractionation, followed by PC cellulose chromatography and affinity chromatography (using Sepharose 4B to which (G)n was covalently bonded). [Schmukler et al. J Biol Chem 250 2206 7975.]... 

Subtilisin (from Bacillus subtilis) [9014-01-1 ] [EC 3.4.21.62]. Purified by affinity chromatography using 4-(4-aminophenylazo)phenylarsonic acid complex to activated CH-Sepharose 4B. [Chandraskaren and Dhai Anal Biochem 150 141 7955]. 

The protein can be further purified by hydrophobic interaction chromatography on a column of Butyl Sepharose 4 Fast Flow (Pharmacia elution with decreasing concentration of (NH4)2S04 starting at 1.5 M), and gel filtration on a column of Superdex 200 Prep (Pharmacia Inouye et al., 2000). 

Step 2. Hydrophobic interaction chromatography on a column of Phenyl-Sepharose CL-4B. The sample was adsorbed on the column in the basic buffer containing 0.5 M (NH SC. The photoprotein adsorbed was first washed with the same buffer, then eluted with the basic buffer. 

Holzman, T. F., and Baldwin, T. O. (1982). Isolation of bacterial luciferases by affinity chromatography on 2,2-diphenylpropylamine-Sepharose phosphate-mediated binding to immobilized substrate analogue. Biochemistry 21 6194-6201. 

Size exclusion chromatography has been used to analyse the size distribution of liposomes. For example, SUV can be separated from MLV, which elute in the void volume, by using a Sepharose 4B gel. 

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