Proteus IMAC guide glossary

affinity chromatography - chromatographic separation based on a specific interaction between an immobilized ligand and a binding site on a macromolecule.

baculovirus – a virus vector for expression of recombinant proteins in insect cells.

bed volume - the total volume occupied by the chromatographic packed bed. It is also referred to as the column volume or CV.

chaotropic agent - a molecule which interferes with hydrophobic interactions by disrupting the ordered structure of water molecules. Examples include urea and guanidine.

chelating agent – a compound such as EDTA or EGTA that is able to combine with a metal ion to form a structure with one or more rings.

cleared lysate – the soluble cell extract after the cell debris and other particulates have been removed by centrifugation.

expression vector – a cloning vector intended for the foreign gene to be expressed in the host organism.

french pressure cell – a device that uses high shear forces to rupture microbial cells. The suspension is poured into a chamber, which is closed at one end by a needle valve and at the other end by a piston. Pressures of up to 16,000 lb/in2 are applied by a hydraulic press against a closed needle valve. When the desired pressure is attained, the needle valve is fractionally opened to marginally relieve the pressure. The cells subsequently expand and rupture, thereby releasing the cellular components through the fractionally open valve. 

freeze-thawing – a method that is sometimes used to break open cells by successive periods of slow freezing and thawing. Ice crystals are generated during the freezing stage, which disrupt the cells when they melt during thawing. The method, however, is slow and releases a limited amount of subcellular components.

his - a 3 letter symbol for L-histidine

his-tag – a permanent affinity tag engineered into the expression vector upstream or downstream of the gene of interest to facilitate the purification of the recombinant protein. The His-tag doesn’t normally have any effect upon the protein structure or function, it comprises 6 x Histidine residues (Hexahistidine) and has a molecular weight of 0.7- 0.9 kDa

immobilized - bound to a surface, usually through covalent linkages.

inclusion bodies – quite a lot of proteins form insoluble crystalline aggregates known as inclusion bodies when they are expressed at high levels inside bacteria. The proteins can be solubilized using denaturants such as 8 M urea or 6 M guanidine hydrochloride.

ion exchange chromatography - chromatographic separation based on different charge properties of macromolecules.

isoelectric point - the pH at which the protein has no net charge.

lysozyme – an enzyme than hydrolyzes β-1,4-linkages between N-acetylmuramic acid and 2-acetamido-2-deoxy-Dglucone in peptidoglycan heteroploymers of prokaryotic cell walls. An example is egg white lysozyme and this enzyme is used to disrupt cells in order to liberate expressed proteins. 1 mg/ml lysozyme is normally added to E.coli cells in lysis buffer and incubated for 30 min to aid cell disruption. The pH optimum for lysozyme is pH 9.2 (Davies et al 1969).

metal chelate affinity chromatography – a form of affinity chromatography where a suitable chelator such as iminodiacetic acid is cross-linked via long stable hydrophilic spacer arm to a matrix such as agarose. The resin is then saturated with an appropriate metal ion, which then has a high affinity for peptidic metal chelates such as poly His-tags.

recombinant protein – a protein coded for by a cloned gene which has often been modified to increase the expression of that protein or to alter the properties of the protein.

sonication – this technique uses ultrasonic energy to generate high transient pressures that are believed to disrupt the cells.

truncate - terminate prematurely or to shorten by cutting.