TITLE: NEBExpress Ni Spin Column Reaction Protocol (NEB #S1427)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Ni resin can be used for the purification of His-tagged fusion proteins under native or denaturing conditions</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>The binding capacity of NEBExpress</span><span style = "vertical-align:super;">®</span><span> Ni Spin Columns is ≥ 1 mg per column. The binding capacity can 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.</span></li><li style = "counter-reset:ol0;list-style-type:disc;">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.</li></ul></div></div>

[STEPS]
?. [Buffer Preparation for Ni Spin Columns]
Prepare buffers for Ni Spin Columns as follows:   ABCD1Lysis/Binding Buffer:

20 mM sodium phosphate, 
300 mM NaClWash Buffer:

20 mM sodium phosphate,
 300 mM NaCl,
 5 mM ImidazoleElution Buffer:

20 mM sodium phosphate,
300 mM NaCl,
500 mM Imidazole22X IMAC Buffer7.5 ml5.0 ml2.5 ml32M Imidazole-0.025 ml1.25 ml4H2O7.5 ml5 ml1.25 ml5Total15.0 ml10.0 ml5.0 ml
ABCD1Lysis/Binding Buffer:

20 mM sodium phosphate, 
300 mM NaClWash Buffer:

20 mM sodium phosphate,
 300 mM NaCl,
 5 mM ImidazoleElution Buffer:

20 mM sodium phosphate,
300 mM NaCl,
500 mM Imidazole22X IMAC Buffer7.5 ml5.0 ml2.5 ml32M Imidazole-0.025 ml1.25 ml4H2O7.5 ml5 ml1.25 ml5Total15.0 ml10.0 ml5.0 ml
Notes:When the recommended protocol is followed, each isolation requires the following volumes of buffer:  ,    and  . An excess volume of each concentrated buffer is provided for preparation of cell lysates or to optimize the concentration of imidazole in the washes.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 10 mM (≥10 mM can reduce the isolated yield but may result in increased purity). imidazole concentration should be determined empirically.Refer to the Chemical Compatibility table prior to including other additives.
?. [Buffer Preparation for Ni Spin Columns]
To prepare buffers for Ni Spin Columns under Native Conditions: bring all three buffers to a final pH of 7.4To prepare buffers for Ni Spin Columns under Denaturing Conditions: bring all three buffers to a final concentration of  or  .
[Urea]
[Guanidine]
?. [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 a 2 µl aliquot of the clarified lysate for SDS-PAGE analysis.
Centrifuge: 12000 34, 15 min
Note: A standard isolation typically employs 0.5 ml of clarified lysate.
?. [Column Preparation]
Remove the bottom tab of the column by twisting, loosen the top cap and place the column in the collection tube provided.
?. [Column Preparation]
Centrifuge column at  to remove the storage buffer, discard the buffer.
Centrifuge: 800 34, 1 min
?. [Column Preparation]
Add  to the column.
[Lysis/Binding buffer]
?. [Column Preparation]
Centrifuge column at , discard the Lysis/Binding buffer.
Centrifuge: 800 34, 1 min
?. [Column Preparation]
Place the column in a new 2 ml microcentrifuge tube.
?. [Lysate Binding]
Add up to  to the column.
[protein sample lysate]
Note: If sample volume is greater than 500 μl multiple applications can be performed; collect the flow through in separate microcentrifuge tubes.
?. [Lysate Binding]
Tap the column to mix the lysate with the resin and allow binding for .
Note: Binding of some His-tagged proteins can be increased with longer mixing times. Cap the column and seal the bottom using the plug provided. Mix end-over-end at   for desired time (typically  - ). Prolonged mixing may result in more non-specific binding.
?. [Lysate Binding]
Centrifuge column at , reserve flow through.
Centrifuge: 800 34, 1 min
?. [Lysate Binding]
Place the column in a new 2 ml microcentrifuge tube.
?. [Column Wash]
Add  to the column and centrifuge at .
[wash buffer]
Centrifuge: 800 34, 1 min
?. [Column Wash]
Repeat wash step twice, collect each wash in a separate 2 ml microcentrifuge tube: Add  to the column and centrifuge at .                                                                                                           (1/2)
[wash buffer]
Centrifuge: 800 34, 1 min
?. [Column Wash]
Repeat wash step twice, collect each wash in a separate 2 ml microcentrifuge tube: Add  to the column and centrifuge at .                                                                                                           (2/2)
[wash buffer]
Centrifuge: 800 34, 1 min
?. [Protein Elution]
Place column in a new 2 ml microcentrifuge tube.
?. [Protein Elution]
Add  to the column. Mix the resin with the elution buffer thoroughly.
[elution buffer]
Note: Elution volume can be reduced to   if a more concentrated protein sample is desired, total target protein yield may be lower.
?. [Protein Elution]
Centrifuge at , save eluted sample.
Centrifuge: 800 34, 1 min
?. [Protein Elution]
Place column in a new 2 ml microcentrifuge tube and repeat elution step. Typically, >90% of the bound protein is eluted following the second elution.
?. [Protein Elution]
Analyze the clarified cell lysate (load), flow through, washes and eluates by SDS-PAGE.