NEBExpress^® Ni Resin Gravity Flow Typical Protocol (NEB #S1428)

Ni resin can be used for the purification of His-tagged fusion proteins under native or denaturing conditions

· The binding capacity of NEBExpress^® Ni Resin is ≥ 10 mg/ml. The binding capacity will vary depending on the size of the target protein, binding conditions and the accessibility of the His-tag. An exact protocol may need to be optimized by the user.

· It is recommended to estimate the expression level of the His-tagged protein of interest by first running a sample on an SDS-PAGE gel.

Recommended Buffers

Lysis/Binding Buffer: 20 mM sodium phosphate, 300 mM NaCl, pH 7.4

Wash Buffer: 20 mM sodium phosphate, 300 mM NaCl, 5 mM Imidazole, pH 7.4

Elution Buffer: 20 mM sodium phosphate, 300 mM NaCl, 500mM Imidazole, pH 7.4

Preparation of Buffers under Denaturing Conditions

1. Bring all three buffers (Lysis/Binding, Wash and Elution Buffers) to a final concentration of 8M Urea or 6M Guanidine.
   
   Notes.
   
   1. Crude lysate should be prepared with a lysis buffer without imidazole. To further minimize contaminants in the eluate, the concentration of imidazole in the wash buffer can be increased to 15 mM (≥15 mM can reduce the isolated yield but may result in increased purity). Imidazole concentration should be determined empirically.
   
   2. Refer to the Chemical Compatibility table prior to including other additives.

Sample Preparation

1. Harvest cells by centrifugation at 4,000 x g for 15 minutes, store the pellet at -20°C or process immediately.
   
   Note: it is recommended to pre-weigh the vessel prior to addition of cell suspension, in order to determine the mass of cell pellet used.
   
   
2. Resuspend cell pellet in Lysis buffer and lyse using method of choice (use approximately 5 mL of lysis buffer per gram of cell paste).
   
   Note: Cells can be lysed by standard methods including sonication, repeated freeze-thaw cycles, French press, etc. Other commercially available lysis reagents can also be used, following manufacturer’s instructions. It is recommended that imidazole be omitted from any lysis buffer.
   
   
3. Centrifuge sample at 12,000 x g for 15 minutes to pellet cellular debris. Remove the clarified protein lysate supernatant and transfer to a new microcentrifuge tube on ice, retain an aliquot of the clarified lysate for SDS-PAGE analysis.

Resin Preparation

1. Gently shake the Ni Resin bottle to completely resuspend the slurry and transfer desired amount to a closed column, allow resin to settle.
   
   
2. Open column and allow storage buffer to flow through.
   
   
3. Equilibrate the resin by adding 5 column volumes (CV) of Lysis/Binding buffer, allow buffer to flow through.

Binding

1. Load protein sample lysate onto the column and allow it to interact with the resin for ≥ 5 minutes.
   
   
2. Collect the flow through in a clean tube. Reserve for SDS-PAGE analysis.
   
   Note: Binding of some His-tagged proteins can be increased with longer incubation times. Column should be placed at 4°C when extending the binding time to maintain activity of the target protein. Prolonged incubation may result in more non-specific binding.

Column Wash

1. Add ≥ 10 CV of Wash buffer to the column, allow buffer to flow through. Flow through can be monitored with absorbance at 280 nm to ensure complete removal of unbound lysate effluent.

2. Collect flow through in fractions and reserve for SDS-PAGE analysis.

Protein Elution

1. Elute fusion protein with Elution buffer.
   
   Note: it is recommended to use a minimum of 3 CV of elution buffer to elute the target protein. Optimal amounts may need to be determined empirically for different substrates.
   
   
2. Collect flow through in fractions and monitor protein elution by measuring the absorbance at 280 nm or by SDS-PAGE.
   
   
3. Analyze the protein sample lysate (load), flow through, washes and eluates by SDS-PAGE.
   
   Note: To remove imidazole for downstream applications use gel filtration (e.g., Zeba ^TM Spin Desalting Columns) or dialysis (e.g. slide-A-Lyzer^R Dialysis Cassettes)

Guidelines for Ni Resin Cleaning

When using a gravity column, the flow rate should be determined prior to cleaning to ensure an accurate contact time with the cleaning agent.

To remove lipoproteins, lipids and proteins strongly bound by hydrophobic interaction follow one of these three methods:

1. Add 1 M NaOH to the column and allow a contact time of 1 to 2 hours, followed by 10 CV of Lysis/Binding Buffer.
   
   
2. Add 30% isopropanol to the column and allow a contact time of 15-20 minutes, followed by 10 CV of water.
   
   
3. Add 0.1 – 0.5% non-ionic detergent in 0.1 M acetic acid to the column and allow a contact time of 1 – 2 hours, followed by 10 CV of water.

To remove proteins bound by ionic interactions

1. Add 1.5 M NaCl to the column and allow a contact time of 10 – 15 minutes. followed by 10 CV of water.

Storage

1. For long-term storage, NEBExpress^™ Ni Resin should be stored in 20% ethanol at 2 – 8°C.

Chemical Compatibility

¹If reagents contain 10 mM EDTA, do not mix the sample and the resin for more than 24 hours before washing and eluting.

²If reagents contain 100 mM EDTA, do not mix the sample and the resin for more than 2 hours before washing and eluting.