New England Biolabs, S1427L, NEBExpress® Ni Spin Columns\n 

Related Categories Amylose Purification (MBP-tag),, Nickel Purification (His-tag),, Affinity Purification, 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 Varies with target, ≥ 1 mg His-tagged fusion protein per column. FAQ Q: How much lysate can be loaded onto a single NEBExpress Ni Spin Column? A: Each column can hold up to 500 µl of lysate per application and provides approximately 1 mg of available binding capacity. The binding efficiency can vary significantly between targets and is dependent on the size of the protein and the accessibility of the His-tag. It is important to estimate the expression level of your target protein, this can be done by running both the lysate and a standard (of known concentration) for quantitation using SDS-PAGE. 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. Q: What is the minimum recommended load volume? A: The minimum recommended load volume is 50 µl per column. Q: Is it necessary to cap the column during each centrifugation step? A: No, centrifugation steps may be carried out without capping the column. Capping the column does reduce the flow rate thereby extending binding time. Q: Why is the liquid not completely removed during the centrifugation step? A: Some centrifuges may vary in centrifugal force. It is also possible that variation in the viscosity of the cleared lysate results in less efficient clearing during centrifugation. Centrifugation time can be increased to 2-5 minutes when necessary. Q: I’ve prepared my lysate with too much buffer and the target is very dilute, can I apply more lysate by repeating the load steps? A: Yes, if the lysate volume is larger than 500 μl, multiple applications to the same column can be performed. Optimal target binding to the resin is related to the concentration of the target, so in some cases, where significant dilution has occurred, it may be necessary to prepare a more concentrated lysate for optimum binding of the target protein. Q: How can I reduce contaminating proteins in a Ni spin column protein purification? 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 washes) 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. 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 5mM imidazole? A: NEBExpress™ Ni Spin Columns utilize a highly selective, proprietary resin that unlike conventional Ni-NTA, requires less imidazole in the purification. Nickel ions are immobilized on the matrix surface via a proprietary ligand that is highly uniform and stable and offers excellent resistance to a wide range of chemicals, including NaOH, EDTA, DTT and β-ME.