Proteins, affinity chromatographic purification

Kumar A, Bansal V, Andersson J, Roychoudhury PK, Mattiasson B (2006), Super-macroporous cryogel matrix for integrated protein isolation - immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line, J. Chromatogr. A 1103 35-42. 

Nevens, J. R., Mallia, A. K., Wendt, M. W., and Smith, P. K. (1992). Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein. ]. Chromatogr. 597, 247-256. 

Intracellular Recombinant Production and Affinity Chromatographic Purification of Proteins... 

Table 1 Comparison of the recombinant production and affinity chromatographic purification of GFP from B. megaterium [30]. Different affinity tag fusion forms of GFP were produced in II. megaterium WH323. Purification was performed using affinity chromatography. Amounts of purified GFP-Strep were determined using a Bradford protein assay kit (Bio-Rad Munich Germany) and BSA (Perbio Rockford USA) as standard. Amounts of purified GFP-His, His-TEV-GFP, Strep-Xa-GFP and Strep-TEV-GFP were calculated via their relative fluorescence per mg protein...

Hematoporphyrin (3,3 -[7,12-bis-(l-hydroxyethyl)-3,8,13,17-tetramethyl-porphyrin-2,18-diyl]-dipropionic acid) [ 14459-29-1 ] M 598.7, pKest 4.8. Purified by dissolving in EtOH and adding H2O or Et20 to give deep red crystals. Also recrystd from MeOH. UV has A, ,ax 615.5, 565, 534.4 and 499.5nm in 0.1 N NaOH, and 597, 619, 634,653, 683 and 701nm in 2 N HCl. [Falk Porphyrins and Metalloporphyrins Elsevier, NY, p 175 1964.] It is used in the affinity chromatographic purification of Heme proteins [Olsen Methods Enzymol 123 324 1986.] The 0-methyl-dimethyl ester has m 203-206° (from CHCh-MeOH) and the 0,0 -dimethyl-dimethyl ester has m 145° (from CHCh-MeOH). [Paul Acta them Scand 5 389 1951.]... 

TABLE 3.6 Examples of ligands suitable for affinity chromatographic purification of proteins... 

Affinity chromatographic purification of an 327 arabinogalactan protein from Gladiolus... 

Affinity chromatography of Ruta graveolens C>-methyltransferases 336 Affinity chromatographic purification of folate-binding protein in 355 cow s whey... 

Affinity chromatographic purification of histamine-binding proteins from human serum... 

Affinity chromatographic purification of kallikrein from human serum 378 Affinity chromatographic isolation of thiamine-binding protein from 379 chicken egg-white... 

Affinity chromatographic purification of the mixed-type putative 382 acetylcholine receptor from wild and mutant strains of house flies Affinity chromatography of cholinergic proteins... 

Affinity chromatographic purification 248 of C-reactive protein from ascites fluid... 

Prepare two chitin column for affinity-chromatographic purification of expressed fusion protein. Therefore, fill slurry of chitin beads in a column and allow them to decant (final bed volume 15 mL). Wash chitin beads with water (20 bed volumes) and equilibrate with column buffer containing 3 M urea (10 bed volumes). 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

Chromatography is one of the most important tools in protein purification. Chromatographic purification techniques include affinity chromatography (AC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and gel filtration (GF). These techniques separate proteins according to differences in specific protein properties. The protein property used for separation, the attributes of each technique, and its suitability for different purification steps are summarized in Table 32.7. 

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