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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>NEBExpress® Ni Resin is an affinity matrix for the isolation and purification of polyhistidine-tagged (His-ta</span><span style = "font-style:italic;">g</span><span style = "font-style:italic;font-style:italic;">g</span><span style = "font-style:italic;">ed</span><span style = "font-style:italic;font-style:italic;">)</span><span style = "font-style:italic;"> fusion proteins</span><span>. It is inte</span><span style = "font-style:italic;">nde</span><span>d fo</span><span style = "font-style:italic;">r </span><span>use in gravity</span><span style = "font-style:italic;"> or </span><span>pressure flow</span><span style = "font-style:italic;"> co</span><span>lumns, a</span><span style = "font-style:italic;">nd</span><span> 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.</span></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]
?. [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 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]
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 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.
?. [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
?. [Pressurized Column Purification]
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 (10cm bed height, 25°C, low viscosity buffer).
NEBExpressTM Ni Resin is suitable for large scale purifications using a pressurized FPLC system.
?. [Pressurized Column Purification]
When running the column purification, the maximum pressure for the resin is 3 bar (0.3 MPa).
?. [Guidelines for Resin Cleaning]
To remove lipoproteins, lipids and proteins strongly bound by hydrophobic interaction follow one of these three methods: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.Add 30% isopropanol to the column and allow a contact time of 15-20 minutes, followed by 10 CV of water.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.
When using a gravity column, the flow rate should be determined prior to cleaning to ensure an accurate contact time with the cleaning agent.
?. [Guidelines for Resin Cleaning]
To remove proteins bound by ionic interactions
?. [Storage]
For long-term storage, NEBExpress Ni Resin should be stored in 20% ethanol at 2 – 8°C.