Why we use immobilized metal affinity chromatography?
Immobilized-metal affinity chromatography (IMAC) is a common technique used to purify recombinant proteins fused to a short peptide affinity tag. Low pH will also elute, but it can sometimes denature the target protein.
How does immobilized metal affinity chromatography work?
Immobilized metal affinity chromatography (IMAC) is a specialized variant of affinity chromatography where the proteins or peptides are separated according to their affinity for metal ions that have been immobilized by chelation to an insoluble matrix.
What is an IMAC column?
Nickel columns are used for immobilized metal affinity chromatography (IMAC) for the purification of recombinant proteins with a polyhistidine tag on either terminus. A recombinant protein with a 6xHis tag has a high affinity for nickel, whereas most other proteins will either bind with low affinity, or not at all.
How does one typically elute protein in immobilized metal ion chromatography?
Elution (desorption) of the target protein is achieved by protonation (using elution buffers with lower pH or lowering pH gradients) and ligand exchange of the metal ion by a stronger chelator (such as EDTA). Divalent ions, such as the transition metals Fe2 +, Co2 +, Ni2 +, Cu2 +, and Zn2 + are most commonly used.
Why are immobilized enzymes more stable?
An immobilized enzyme may show selectively altered chemical or physical properties, and it may provide a better environment for the enzyme activity. Therefore, immobilized enzymes are often more stable than free enzymes in a solution.
Which metal is used in chromatography?
Stainless steel and glass are the usual materials for packed columns and quartz or fused silica for capillary columns. Gas chromatography is based on a partition equilibrium of analyte between a solid or viscous liquid stationary phase (often a liquid silicone-based material) and a mobile gas (most often helium).
What is Ni-NTA column?
Ni-NTA Agarose is a nickel-charged affinity resin that can be used to purify recombinant proteins containing a polyhistidine (6xHis) sequence. Ni-NTA uses the chelating ligand nitrilotriacetic acid (NTA) coupled to a cross-linked 6% agarose resin that is suitable for use in batch and gravity flow applications.
What are the advantages of immobilized enzymes?
A main advantage of immobilized enzymes is that they can be reused, as they typically are macroscopic catalysts that can be retained in the reactors. 2. Soluble enzymes can contaminate the product, and their removal may involve extra purification costs.
What is the stationary phase in IMAC?
The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups (e.g., His residues) in peptides (e.g., 3-7) and on protein surfaces (8-13).
Immobilized metal affinity chromatography (IMAC) is a protein separation method based on the interaction between proteins in solution and transition metal ions fixed to a solid support [1]. The specificity of the separation is determined by the occurrence and position of metal-coordinating residues on the protein surface.
What makes a metal chelate in a chromatography?
Electron-donor atoms (or nucleophiles such as N, S, and O) present in the chelating compounds attached to the chromatographic support are capable of forming metal chelates.
How are affinity resins used in chromatography?
Affinity resins are composed of a macroporous chromatography matrix to which the specific ligand is attached. The pore size of the matrix must be large enough to provide room for the, often bulky, ligand and to provide free access to the ligand for the interacting macromolecule.
How to increase the capacity of HITRAP chelating hp?
To increase capacity use several HiTrap Chelating HP columns (1 mL or 5 mL) in series (note that back pressure will increase) or, for even larger capacity, pack Chelating Sepharose Fast Flow into a suitable column (Appendix 3, Column packing and preparation ).