New England Biolabs, S1428S, NEBExpress® Ni Resin

Related Categories Amylose Purification (MBP-tag),, NEBExpress MBP Fusion and Purification System,, Bacterial E. coli Protein Expression, Applications MBP Affinity Tag,, Fusion Protein Cleavage,, Pull Down Assays, Specification Storage Buffer 20% Ethanol Support Matrix Spherical, agarose based microparticles ranging in size from 10-100 μm. Binding Capacity 1 ml of NEBExpress™ Ni Resin will bind ≥ 10 mg of His-tagged fusion protein. FAQ Q: Can NEBExpress® Ni Resin be used in large scale, pressurized purification systems such as FPLC? A: Yes, NEBExpress® Ni Resin is an excellent choice for large scale purification in these automated systems. The following guidelines are recommended for use in an automated, pressurized system: Columns should be packed at a constant pressure of approximately 1 bar (0.1 MPa) or a packing flow velocity of approximately 400 cm/h (10 cm bed height, 25oC, low viscosity buffer). The maximum pressure the resin can withstand is 3 bar (0.3 MPa). Q: Does NEBExpress Ni Resin need to be regenerated with nickel after cleaning? A: No, the resin is comprised of a highly stable Ni matrix which minimizes nickel ion leakage and eliminates the need for nickel regeneration even after repetitive Clean-in-Place (CIP) cycles. Refer to the typical reaction protocols for recommended CIP procedures. Q: How can I reduce contaminating proteins in a protein purification when using Ni Resin? A: Add up to 10 mM imidazole to the Lysis buffer to prevent contaminants from binding (optimal imidazole concentrations must be determined empirically). Employ extra wash steps (e.g., up to 5 additional column volumes) with the recommended wash buffer. Increase the imidazole concentration in the wash buffer (test by increasing in 5 mM increments up to 20 mM); higher concentrations of imidazole can result in decreased overall yield of the target protein. The optimal concentration of imidazole will be target/impurity dependent and will therefore require optimization. If you suspect contaminants are associated with the His-tag fusion protein, add 10 mM β-Mercaptoethanol or 1 mM THP to reduce disulfide bonds. Increase salt and/or detergent concentrations or add ethanol/glycerol to the wash buffer to disrupt non-specific interactions. Contaminants can also arise as truncated forms of the His-tag fusion protein, check for possible internal translation starts (C-terminal His-tag) or premature termination sites (N-terminal His-tag). Avoid protein degradation during the purification by keeping the resin cold (4°C) or by including protease inhibitors. Q: What are the recommendations for pH based elution, as opposed to imidazole? A: For both imidazole and pH based purifications the cell lysis should be done in buffered solution adjusted to pH 7.4, with sodium chloride supplemented up to 0.3 M. Following binding, a wash buffer with a pH of approximately 6.3 is recommended. Elution can be carried out by utilizing a buffer with a pH between 4.5 and 5.2. Q: How can I remove imidazole from a protein sample? A: Imidazole does not typically interfere with downstream applications and therefore removal is optional. Boiling a sample containing imidazole prior to SDS–PAGE may cause acid-labile bonds to hydrolyze. It is instead recommended to incubate the sample at 70°C for 5 min in SDS loading buffer prior to analysis. If it is necessary to eliminate the imidazole, it can be removed by dialysis, ammonium sulfate precipitation, ultrafiltration or by using a size-exclusion desalting column. Q: What are to the advantages of performing purification under denaturing conditions? A: Deciding whether to purify a His-tagged protein under denaturing or native conditions depends on protein location and solubility, accessibility of the His-tag and whether or not the biological activity must be retained for downstream applications. Purification under denaturing conditions is most commonly employed when the tertiary structure of the native protein sequesters the His-tag, rendering purification under native conditions inefficient, or to solubilize inclusion bodies. Denaturing conditions can also mitigate the activity of proteolytic enzymes, reducing the risk of degradation of the target protein throughout the purification. Q: How can I determine if my target protein remains bound to the resin? A: Add 200 µl of SDS-PAGE loading buffer to the column, pipette up and down and remove an aliquot of the resin from the spin column. Incubate at 70°C for 5 minutes to release any protein that remains on the resin following elution. Load and analyze proteins by SDS-PAGE. Most proteins, regardless of whether they bind to the nickel or to the agarose-bead itself, will be recovered by this procedure. Q: Why is imidazole not necessary in the lysis/binding buffer? Why does the wash buffer contain only 5 mM imidazole? A: NEBExpress Ni resin utilizes a highly selective, proprietary resin that requires less imidazole throughout the purification in comparison to conventional Ni NTA based resins. The optimal concentration of imidazole will be target/impurity dependent and will therefore require optimization. Q: Why is my protein precipitating on the column? A: It is possible that the protein forms aggregates, or that the temperature is too low. Precipitation can be reduced by running the purification at room temperature, performing batch mode binding to reduce high local protein concentration, and/or adding solubilization reagents to working buffers (i.e. 0.1% Triton X-100 or Tween20, < 10 mM β-Mercaptoethanol, up to 2M NaCl or cofactors such as Mg2+). Q: What gravity column do you recommend? A: We recommend the 2.5 cm (internal diameter) Econo Column line from Bio-Rad or the Kontes Flex-Column. Column sizes (internal diameter and length) can be scaled up or down and should be based on the amount of resin needed to isolate your protein of interest in sufficient yields. Q: Will extended binding increase the yield in the eluate using NEBExpress® Ni Spin Columns? A: In some cases, extending the binding step can result in significantly increased binding of the target protein. Cap the column and seal the bottom using the plug provided. Mix end-over-end at 4°C for desired time (typically 5-15 minutes but can be extended overnight if necessary). However, keep in mind that prolonged mixing may also result in increased non-specific protein binding.