Affinity chromatography purification

Fig. 21 Chitin binding of 6xHis-tagged resilin with chitin-binding domain (6 H-resChBD) as compared to 6xHis-tagged resilin without chitin-binding domain (6 H-res). T total protein after affinity chromatography purification, B bound protein eluted from chitin beads, UB unbound protein. Reproduced from [187] with permission from The American Chemical Society, copyright 2009...

Kariluoto, M. S., Vahteristo, L. T, Piironen, V. 1. (2001). Applicability of microbiological assay and affinity chromatography purification followed by high-performance liquid chromatography (HPLC) in studying folate contents in rye. J. Sci. Food Agric., 81, 938-942. 

Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography purification up to 40 000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 kDa and an isoelectric point of 7.9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it in a number of characteristics. 

Skorey, K.I., N.A. Johnson, G. Huyer, and MJ. Gresser. 1999. A two-component affinity chromatography purification of Helix pomatia arylsulfatase by tyrosine vanadate. Prot. Expr. Purif. 15 178-187. 

The specific activity of the enzyme was determined in 0.15M lactose solution (0.02M phosphate buffer, pH 7-0). The activity of the enzyme was expressed in terms of units of activity per mg of enzyme. A unit of activity was defined as a p mole of glucose produced per minute. The soluble lactase activity following affinity chromatography purification was 37-1 units/mg. This represented a 4 fold increase in catalytic potency over the specific activity of the crude enzyme preparation (8.9 units/mg). 

Figure 2 Cause-and-effect (fish-bone) diagram for affinity chromatography purification.

Willoughby N.A., Kirschner T., Smith M.P., Hjorth R. and Titchener-Hooker N.J. 1999. Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker s yeast using an expanded bed adsorption system, J. Chromatogr. A, 40, 195-204. 

Cyanobacterial Hepatotoxin Microcystins Affinity Chromatography Purification... 

An essentially pure extracellular glycoprotein proteinase inhibitor was isolated from the latex of green fruits of papaya by a single affinity chromatography purification step. An immobilized trypsin-Sepharose CL 4B column was prepared according to the manufacturer (Amersham Pharmacia Biotech), which provided elaborated bulletins for the preparation procedures and applications of these affinity gel columns. Latex extract was applied to the column after equilibration with 20 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl and 0.05 M CaCli. The column was extensively washed with the same buffer until ultraviolet (UV) absorbance became undetectible. The bound trypsin inhibitor was eluted with 0.02 M HCl and recovered by lyophilization after dialysis against water. 

High detergent concentrations can be used for the resolubilization screens. Concentrations, as well as type of detergent, may be modified during subsequent affinity chromatography purification steps. 

Allen RH, Mehlman CS. Isolation of gastric vitamin B12 binding-protein using affinity chromatography. Purification and properties of human intrinsic factor. J Biol Chem 248 3660-3680, 1973. 

Purification of jS-o-galactosidase by affinity chromatography Isolation of glycogen phosphorylase B by affinity chromatography Purification of guanine aminohydrolase by affinity chromatography... 

Purification of oestradiol receptor of calf uterus by affinity chromatography Purification of collagenase by affinity chromatography isolation of anti-haptoglobin antibodies by immunoadsorption Removal of interspecies reactivity from anti-(immunoglobulin G) sera (human or rabbit) Investigation of the properties of the immobilized IgG... 

Direct affinity chromatography purification of protein interactors in relevant cells/tissues... 

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