|Product||Cat. No.||Amount||Price (EUR)||Buy / Note|
|Low Density Nickel Agarose||AC-304-25||25 ml||145,00||Add to Basket/Quote Add to Notepad|
|Low Density Nickel Agarose||AC-304-100||100 ml||435,00||Add to Basket/Quote Add to Notepad|
|Low Density Nickel Agarose||AC-304-500||500 ml||1.800,00||Add to Basket/Quote Add to Notepad|
For research use only!
Shipping: shipped at ambient temperature
Storage Conditions: store at 4 °C
do not freeze
Shelf Life: 12 months
Form: supplied as a suspension in 20 % ethanol
Metal-containing affinity resins are designed for batch or column purification of His-tagged proteins.
General Procedure for the Purification of His-tagged Proteins
The following procedure describes the purification of His-tagged proteins under native conditions. The affinity of the protein to the resin will depend on first at all the chemistry of the resin (number of chelate groups, chelating metal), second the accessibility of the His-tag, and third pH and composition of the buffer.
1. Removal of the storage solution
Wash the beads with 5 - 10 column volumes of distilled water to eliminate the storage solution.
Please note: In case of Metal Free Agaroses (AC-301, AC-302), the column must be loaded with metal prior to use, as described in the following steps:
Equilibrate the column with 5 - 10 column volumes of binding buffer.
The choice of binding buffer depends on the particular properties of the protein as well as on the type of chelate used. The most frequently used buffers are acetate (50 mM) or phosphate (10 - 150 mM). The pH of binding buffers is usually close to neutral pH (7.0 - 8.0), but can vary over the range 5.5 - 8.5. To avoid ionic interchange, add 0.15 - 0.5 M of NaCl.
Please note: In some cases addition of imidazole (10 - 40 mM) to the binding buffer will increase the selectivity of protein binding. Usage of high purity imidazole will avoid affecting OD280 measurements. Avoid the presence of chelating agents such as EDTA or citrate during the complete purification process.
3. Sample loading
Once the resin is equilibrated, the sample containing the tagged protein for purification is applied. In some cases, a slight increase of contact time may facilitate binding.
Please note: Binding capacity can be affected by several factors, such as sample concentration, binding buffer or the flow rate during sample application.
Wash the resin with the binding buffer until OD280 nm reaches baseline level.
Elution of the purified protein can be performed in several ways:
Recombinant proteins often form insoluble inclusion bodies which have to be rendered soluble by a purification under denaturing conditions, e.g. by using 7 M urea or 6 M guanidine chloride during the purification process.
The following procedure describes how a column is packed from bulk affinity resins:
Linear flow rate: 26 cm/h
Recommendes flow rate: 0.5 - 1.0 ml/min
Max. pressure: 2.6 psi (0.18 bar)
Regeneration of Chelating Resins
Binding capacity of chelating resins may decrease after several cycles of usage, because some protein may have been retained. To revert binding capacity to the starting state, regeneration may be necessary. The regeneration procedure will completely remove the metal and therefore also any retained protein.
In general, column regeneration is always recommended when beginning to purify a new protein. When continuing with the same protein, it is recommended to perform a regeneration when an appreciable diminution of the yield is observed. The frequency of these stages varies with the protein and the conditions used.
Gasset-Rosa et al. (2008). Negative regulation of pPS10 plasmid replication: origin pairing by zipping-up DNA-bound RepA monomers. Mol. Microbiol. 68:560.
Giraldo (2007). Defined DNA sequences promote the assembly of a bacterial protein into distinct amyloid nanostructures. Proc. Natl. Acad. Sci. USA 104:17388.
Bastida et al. (2003) In Vivo Chaperone-Assisted Folding of 1,6-Fucosyltransferase from Rhizobium sp. Chem Bio Chem 4:531.
Yip et al. (1994) Immobilized Metal Ion Affinity Chromatography. Molecular Biotechnology 1:151.
Anspach et al. (1994). Silica-based metal chelate affinity sorbents. I. Preparation and characterization of iminodiacetic acid affinity sorbents prepared via different immobilization techniques. Journal of Chromatography A 672:35.
Porath (1992) Immobilized Metal Ion Affinity Chromatography. Protein Expression and Purification 3:263.
Porath et al. (1991) Cascade-mode multiaffinity chromatography. Fraction of human serum protein. Journal of Chromatography 550:751.
Hemdan et al. (1985) Development of immobilized metal affinity chromatography. II: Interaction of amino acids with immobilized Nickel Iminodiacetate. Journal of Chromatography 323:255.
Porath et al. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598.