What is Ni-NTA chromatography?
What is Ni-NTA chromatography?
Ni-NTA Agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a His tag. Histidine residues in the His tag bind to the vacant positions in the coordination sphere of the immobilized nickel ions with high specificity and affinity. Cleared cell lysates are loaded onto the matrices.
How do you clean Ni-NTA resin?
Wash the resin with 10 column volumes of distilled water to eliminate the isopropanol. To eliminate lipids, wash in batch for two hours with a solution 0.5% of non-ionic detergent in 0.1 M acetic acid. Rinse the detergent with ethanol 70% (approximately 10 column volumes).
What are the two methods of elution of protein from Ni-NTA?
Recombinant protein is usually eluted from an Ni column with a high concentration of imidazole. Other elution methods include lowering pH, so that the histidines become protonated and no longer have affinity for the nickel resin, or using strong chelating agents such as EDTA and EGTA.
What is EDTA and NTA?
Ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA) are two aminocarboxylic acids known to increase metal uptake by plants ( 1−3). Knowledge of the stability of metal-chelate complexes and free chelate ions in nutrient solutions is lacking due to the difficulty of analysis.
How do you clean Ni-NTA beads?
Ni-NTA matrices are stable under a wide variety of conditions and need not be refrigerated, except to inhibit growth of microorganisms for long-term storage. After use they should be washed for 30 minutes with 0.5M NaOH.
What is IMAC protein purification?
Immobilized metal affinity chromatography (IMAC) IMAC is a widely-used method for rapidly purifying polyhistidine affinity-tagged proteins, resulting in 100-fold enrichments in a single purification step. The chelators most commonly used as ligands for IMAC are nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA).
How much resin do you use for protein purification?
To set up protocol to purify a new protein, it is recommended to start with the ratio of 1 mL resin for every 20 mL of culture supernatant or lysate. It also helps to estimate the amount of target protein in the sample and adjust it to approximately 0.5 mg/mL.
What is Ni-NTA resin used for?
The Ni -NTA Resin is specifically designed for the purification of recombinant proteins fused to the 6 x histidine (6XHis) tag expressed in bacteria, insects, and mammalian cells.
How do you purify proteins with Ni NTA?
Proteins can be purified under native, denaturing, or hybrid conditions using the Ni-NTA Agarose. Proteins bound to the resin are eluted with low pH buffer or by competition with imidazole or histidine. The resulting proteins are ready for use in target applications.
How to regenerate the Ni-NTA column?
Column regeneration should be performed when a different protein is being isolated or when there is a significant loss in the yield of protein. If the Ni-NTA Resin loses its blue color the column needs recharging. Wash the resin with 5 column volumes of a solution 20mM sodium phosphate supplemented with 0.5M NaCl, 50mM EDTA at pH 7.0.
Should I purify the target protein under native or denaturing conditions?
The choice whether to purify the target protein under native or denaturing conditions depends on protein location and solubility, the accessibility of the His tag, and the desired downstream application.
