Preparative chromatography affinity

For the SAXS studies a CBH II sample was prepared by affinity chromatography from r. reesei QM 9414 to give the enzyme in a homogeneous form 27. In SDS-PAGE the protein had a size of 58 kDa and the isoelectric point was 4.9. Glycosy-lation was estimated as 8 to 18 % 36. The molar absorptivity at 280 nm was 75 000 M xm To obtain the core protein partial proteolytic hydrolysis with papain was per-... 

Thiophilic chromatography depends on the binding of proteins to sulphur atoms contained in a particular chemical context. During the development of methods for the preparation of affinity supports... 

Papain. Papain was first isolated in crystalline form from fresh latex by Balls et al. 22), but it is more conveniently isolated from commercially available dried latex by the procedure of Kimmel and Smith 23). Papain prepared by this procedure, however, is not fully active see Activation and Inhibition below) and usually contains only 0.5 mole SH per mole of protein. Fully active papain containing I mole SH per mole of protein may be prepared by affinity chromatography on a column of an inhibitor, Gly-Gly-Tyr (Bzl)-Arg, covalently linked to agarose 24). Two other techniques which have been used to purify papain involve... 

Probably the most significant advance in column chromatography over the last five years has been the emergence of affinity chromatography from the state of a laboratory curiosity to that of a widely used standard separation technique. Although the fundamental principles of affinity chromatography can be found in the literature as early as 1910 [1] and 1916 [2], it is only since the late 1960 s that real advancement has been made in this field. To a large extent these advances are due to the development of better techniques for the preparation of affinity media. 

The choice of support in the preparation of affinity media is of critical importance. A wide range of materials is available and the continued interest of commercial companies in chromatography ensures that new media are developed. The major requirments of a support for use in the preparation of affinity media are ... 

IgG in antiserum prepared as described above is purified by affinity chromatography. To prepare the affinity column, conjugate the antigen (component I or II) to Affi-Gel 15 (Bio-Rad Laboratories) according to the manufacturer s Instruction Manual. 

Without doubt, concanavalin A (Con A) is the most celebrated and has proven to be one of the most useful of the plant lectins. Its physical chemical properties and carbohydratebinding properties are well documented in previous reviews [3,8]. Suffice it to note that it was first isolated and crystallized by Sumner and Howell in 1936, who showed it to require metal ions for its activity [74]. By virtue of its interaction with branched a-D-glucans, it is readily prepared by affinity chromatography on crossed-linked dextran (Sephadex) [75, 76]. A homotetramer at pH 7 (subunit M, = 26 500 Da) of Con A has been sequenced [77] and its crystal structure determined both in its native form [38,39] and complexed with methyl a-D-mannopyranoside [40] and Man(al-3)[Man(al-6)]Man[49]. 

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