Sepharose

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. 

In preparing any of the above for use in columns, the dry powder is evacuated, then mixed under reduced pressure with water or the appropriate buffer solution. Alternatively it is stirred gently with the solution until all air bubbles are removed. Because some of the wet powders change volumes reversibly with alteration of pH or ionic strength (see above), it is imperative to make allowances when packing columns (see above) in order to avoid overflowing of packing when the pH or salt concentrations are altered. 

Cellex D and other anionic celluloses are washed with 0.25M NaCl/0.25M NaOH solution, then twice with deionised water. This is followed with 0.25M NaCl and then washed with water until chloride-free. The Cellex is then equilibrated with the desired buffer as above. 

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DEAF Sepharose Fast Flow LC DEAF Sepharose Fast Flow HC DEAF. Spherodex M DEAF. Spherosil M DEAF. Trisacryl Plus M Toyopead DEAE-650 (M) Fractogel EMD DMAE-650 (M) Fractogel EMD DEAE-650 (M) Q Sepharose Fast Flow QMA Spherodex M QMA Spherosil M QMA Trisacryl Plus M Toyopead QAE-550 C SP Sepharose Fast Flow SP Sepharose High Performance SP Sepharose Big Bead SP Trisacryl Plus M Toyopead SP-650 M Fractogel EMD SO -650 M... 

An example of a size-exclusion chromatogram is given in Figure 7 for both a bench-scale (23.5 mL column) separation and a large-scale (86,000 mL column) mn. The stationary phase is Sepharose CL-6B, a cross-linked agarose with a nominal molecular weight range of 5000-2 x 10 (see Fig. 6) (31). 

Fig. 7. Chromatograms of size-exclusion separation of IgM (mol wt = 800,000) from albumin (69,000) where A—D correspond to IgM aggregates, IgM, monomer units, and albumin, respectively, using (a) FPLC Superose 6 in a 1 x 30 — cm long column, and (b) Sepharose CL-6B in a 37-cm column.

Gel-permeation media are extremely versatile and may be used for separation of particles such as vimses (Fig. 11) as well as proteins (34). Separations of proteins and other particles having sizes equivalent to a molecular weight of 40 x 10 are possible using the agar-based Sepharose-type gel. This particular gel has a limited temperature range for operation, however. It melts upon heating to 40°C (34). 

Fig. 11. Separation of P-labeled A, adenovims Type 5, and B, poHovims Type 1 on Sepharose 2B where the column is 2.1 x 56 cm eluent, 0.002 M...

The first observation of the enantioselective properties of an albumin was made in 1958 (28) when it was discovered that the affinity for L-tryptophan exceeded that of the D-enantiomer by a factor of approximately 100. This led to more studies in 1973 of the separation of DL-tryptophan [54-12-6] C22H22N2O2, on BSA immobilized to Sepharose (29). After extensive investigation of the chromatographic behavior of numerous racemic compounds under different mobile-phase conditions, a BSA-SILICA hplc column (Resolvosil-R-BSA, Macherey-Nagel GmvH, Duren, Germany) was... 

ADP-Ribosyl transferase (from human placenta) [9026-30-6]. Purified by making an affinity absorbent for ADP-ribosyltransferase by coupling 3-aminobenzamide to Sepharose 4B. [Burtscher et al. Anal Biochem 152 285 1986.]... 

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]. 

Ceruloplasmin (from human blood plasma) [9031-37-2] Mr 134,000. This principle Cu transporter (90-90% of circulating Cu) is purified by precipitation with polyethylene glycol 4000, balchwise adsorption and elution from QAE-Sephadex, and gradient elution from DEAE-Sepharose CL-6B. Ceruloplasmin... 

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]. 

Purified by ammonium sulfate pptn, then fractionated on Sephadex G-75 column, applied to a Blue Sepharose column and eluted with ImM dihydrofolate. [Al Rubeai and Dole Biochem J 235 301 1986.]... 

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]. 

Pertussis toxin (from Bordetella pertussis) [70323-44-3J Mr 117,000. Purified by stepwise elution from 3 columns comprising Blue Sepharose, Phenyl Sepharose and hydroxylapatite, and SDS-PAGE [Svoboda et al. Anal Biochem 159 402 1986, Biochemistry 21 5516 79[Pg.557]

Phosphoribosyl pyrophosphate synthetase (from human erythrocytes, or pigeon or chicken liver) [9015-83-2] Mr 60,000, [EC 2.7.6.1]. Purified 5100-fold by elution from DEAE-cellulose, fractionation with ammonium sulfate, filtration on Sepharose 4B and ultrafiltration. [Fox and Kelley J Biol Chem 246 5739 197h, Flaks Methods Enzymol6 158 1963 Kornberg et al. J Biol Chem 15 389 7955.]... 

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.]... 

Protease nexin (From cultured human fibroblasts) [148263-58-5], Purified by affinity binding of protease nexin to dextran sulfate-Sepharose. [Farrell et al. Biochem J 237 707 1986.]... 

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.]... 

Ricin (toxin from Castor bean Ricinus communis) [A chain 96638-28-7 B chain 96638-29-8] Mr -60,000, amorphous. Crude ricin, obtained by aqueous extraction and (NH4)2S04 pptn, was chromatographed on a galactosyl-Sepharose column with sequential elution of pure ricin. The second peak was due to ricin agglutinin. [Simmons and Russell Anal Biochem 146 206 1985.) Inhibitor of protein synthesis. EXTREMELY DANGEROUS, USE EXTREME CARE [instructions accompany product]. 

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