Purification Media, Affinity, Nickel

Recombinant proteins tagged with 6-10 poly Histidines can be purified in one step by ion metal affinity chromatography (IMAC). Several metal ions exhibiting affinity to poly-Histidines are commonly used, Nickel and Cobalt being the most popular. Tagging the proteins may be done at the N-ter or the C-ter by cloning a gene in dedicated expression vectors encoding to the poly Histidines stretch, enabling the purification of the recombinant protein resulted. Nickel Beads are high quality Nickel affinity-resin product, that exhibit high capacity and good selectivity towards His-tagged proteins. Purification with Nickel Beads may be done in a variety of formats, such as gravity-flow columns and small or large-scale batches. The preferred purification strategy is to start by using the native purification conditions and only later on, if required, use denaturing conditions for proteins that are accumulated as inclusion bodies or in cases when the 6xHis purification tag is not exposed in the native form. The amount of actual protein bound can vary with the type and size of protein. Reducing agents such as DTT (Dithiothreitol) and chelating agents such as EDTA (Ethylene diamine tetra acetic acid) and EGTA (Ethylene glycol-bis(beta-amino-ethyl ether)), may be used in the buffers employed in the extraction protocols, but not during the affinity purification itself. Gel filtration or, dialysis is recommended procedures for removal of these agents., , Nickel Beads Specifications:, Matrix: Sepharose CL-4B, Activation method: Oxiran., Chelating group: Iminodiacetic acid, Binding capacity: ~6-9 mg pure (His) 6 -tagged protein (Mr ~ 49 000) per ml, Mean bead size: 40-165 µm, Bead structure: Highly cross-linked spherical agarose, 4%, Max back pressure: 0.3 MPa, 3 bar, Max. flow rates: 4 ml/min/cm2, Recommended flow rate: 1-2 ml/min/cm2, Stability of the matrix: pH 2-11., Storage: 4°C in PBS pH 7.4, 0.1% (w/v) sodium azide or 20% ethanol as preservatives. Storage and Stability: Stable at RT for 1 week