TITLE: NEBExpress Ni Resin Gravity Flow Typical Protocol (NEB #S1428)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">NEBExpress® Ni Resin is an affinity matrix for the isolation and purification of polyhistidine-tagged (His-tagged) fusion proteins. It is intended for use in gravity or pressure flow columns, and batch purifications. NEBExpress Ni Resin is comprised of a highly uniform and stable chemical-tolerant resin, pre-charged with nickel ions on the matrix surface.  It is resistant to a wide range of chemicals, including NaOH, EDTA, DTT and β-Mercaptoethanol.</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Purification of ≥10 mg His-tagged protein per 1 ml of resin</li><li style = "counter-reset:ol0;list-style-type:disc;">Intended for use in gravity or pressure flow columns, and batch purifications</li><li style = "counter-reset:ol0;list-style-type:disc;">High specific binding of His-tagged proteins yielding purities of >95%</li><li style = "counter-reset:ol0;list-style-type:disc;">Strong nickel ion binding provides excellent resistance to EDTA and reducing agents. Compatible with commercially available detergent-based cell lysis reagents</li><li style = "counter-reset:ol0;list-style-type:disc;">Isolation and purification of His-tagged fusion proteins under native or denaturing conditions</li></ul></div></div>

[STEPS]
?. [Guidelines for Ni Resin Cleaning]
To remove lipoproteins, lipids and proteins strongly bound by hydrophobic interaction follow one of these three methods:Add  to the column and allow a contact time of  to , followed by 10 CV of Lysis/Binding Buffer.Add 30% isopropanol to the column and allow a contact time of -, followed by 10 CV of water.Add 0.1 – 0.5% non-ionic detergent in  to the column and allow a contact time of  – , followed by 10 CV of water.
When using a gravity column, the flow rate should be determined prior to cleaning to ensure an accurate contact time with the cleaning agent.
[NaOH]
[acetic acid]
?. [Guidelines for Ni Resin Cleaning]
To remove proteins bound by ionic interactionsAdd  to the column and allow a contact time of  – . followed by 10 CV of water.
[NaCl]
?. [Storage]
For long-term storage, NEBExpress Ni Resin should be stored in 20% ethanol at  – .
2 °C
8 °C
?. [Preparation of Buffers under Denaturing Conditions]
If continuing with Denaturing conditions: Bring all three buffers (Lysis/Binding, Wash and Elution Buffers) to a final concentration of   or .
[Urea]
[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]
Harvest cells by centrifugation at , store the pellet at  or process immediately.
Centrifuge: 4000 34, 15 min
-20 °C
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.
?. [Sample Preparation]
Resuspend cell pellet in Lysis buffer and lyse using method of choice (use approximately  per ).
[lysis buffer]
[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.
?. [Sample Preparation]
Centrifuge sample at  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.
Centrifuge: 12000 34, 15 min
?. [Resin Preparation]
Gently shake the Ni Resin bottle to completely resuspend the slurry and transfer desired amount to a closed column, allow resin to settle.
?. [Resin Preparation]
Open column and allow storage buffer to flow through.
?. [Resin Preparation]
Equilibrate the resin by adding 5 column volumes (CV) of Lysis/Binding buffer, allow buffer to flow through.
?. [Binding]
Load protein sample lysate onto the column and allow it to interact with the resin for ≥ .
?. [Binding]
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   when extending the binding time to maintain activity of the target protein. Prolonged incubation may result in more non-specific binding.
?. [Column Wash]
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.
?. [Column Wash]
Collect flow through in fractions and reserve for SDS-PAGE analysis.
?. [Protein Elution]
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.
?. [Protein Elution]
Collect flow through in fractions and monitor protein elution by measuring the absorbance at 280 nm or by SDS-PAGE.
?. [Protein Elution]
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 Spin Desalting Columns) or dialysis (e.g. slide-A-Lyzer Dialysis Cassettes)