TITLE: NEBExpress Ni-NTA Magnetic Beads (NEB #S1423)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">NEBExpress</span><span style = "font-style:italic;vertical-align:super;">®</span><span style = "font-style:italic;">Ni-NTA Magnetic Beads can be used for the purification of His-tagged fusion proteins under native or denaturing conditions</span><span style = "font-style:italic;"></span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">The binding capacity of NEBExpress Ni-NTA Magnetic Beads can vary depending on target and binding conditions. We recommend estimating approximately 7.5 mg of available binding capacity per ml (bed volume) of resin, therefore 50 µl of Ni-NTA bead slurry yields approximately 40 µg of purified protein. An exact protocol may need to be optimized by the user. See the FAQs for more information on protocol optimization.

</li><li style = "counter-reset:ol0;list-style-type:disc;">For optimal performance, the amount of beads used should match the approximate amount of His-tagged protein to be captured.

</li><li style = "counter-reset:ol0;list-style-type:disc;">Crude lysate should be prepared with lysis buffer or a buffer in the pH range of 7.0–8.2 supplemented with up to 10 mM imidazole to reduce non-specific binding of proteins.

</li><li style = "counter-reset:ol0;list-style-type:disc;">Magnetic racks may be purchased separately: 6-tube Magnetic Separation Rack (NEB #S1506); 12-tube Magnetic Separation Rack (NEB #S1509); and 96-Well Microtiter Plate Magnetic Separation Rack (NEB #S1511)</li></ul></div></div>

[STEPS]
?. [Buffer Preparation for NEBExpress Ni-NTA Magnetic Beads]
Prepare the following buffers:  ABCD1Lysis/Binding Buffer:
20 mM sodium phosphate,
300 mM NaCl,
10 mM ImidazoleWash Buffer:
20 mM sodium phosphate,
300 mM NaCl,
20 mM ImidazoleElution Buffer:
20 mM sodium phosphate,
300 mM NaCl,
500 mM Imidazole22X IMAC Buffer1.5 ml10 ml1.25 ml32M Imidazole0.015 ml0.2 ml0.625 ml4H2O1.49 ml9.8 ml0.625 ml5Total3.0 ml20 ml2.5 ml
ABCD1Lysis/Binding Buffer:
20 mM sodium phosphate,
300 mM NaCl,
10 mM ImidazoleWash Buffer:
20 mM sodium phosphate,
300 mM NaCl,
20 mM ImidazoleElution Buffer:
20 mM sodium phosphate,
300 mM NaCl,
500 mM Imidazole22X IMAC Buffer1.5 ml10 ml1.25 ml32M Imidazole0.015 ml0.2 ml0.625 ml4H2O1.49 ml9.8 ml0.625 ml5Total3.0 ml20 ml2.5 ml
Notes:Working buffers are sufficient for 10 reactions using the supplied concentrated buffers; volumes can be scaled up for larger reactions

Ni-NTA beads have a high affinity for His-tagged proteins with minimum non-specific binding. Under native conditions, the stringency of binding is modulated by including a low concentration of imidazole in the binding and wash buffers. The optimal concentration of imidazole in the buffers may need to be optimized depending on the affinity of the target protein for the Ni-NTA beads.
?. Move forward with the protocol, using Single Tubes or 96-well Plates:
?. [Bead Equilibration - Single Tubes]
Resuspend the bead slurry by vortexing or mixing.
?. [Bead Equilibration - Single Tubes]
Immediately dispense  to a 1.5 ml tube.
[bead slurry]
?. [Bead Equilibration - Single Tubes]
Add  to the bead slurry and mix briefly.
[binding buffer]
?. [Bead Equilibration - Single Tubes]
Place the tube in a magnetic rack to pellet the beads, remove and discard the supernatant.
?. [Target Protein Binding - Single Tubes]
Add  to the equilibrated beads.
[crude lysate]
?. [Target Protein Binding - Single Tubes]
Incubate for  at  with end over end mixing or with a benchtop shaker at 850 rpm.
0 Room temperature
Note: Beads may adhere to sides or cap of the tubes during mixing. Samples can be spun briefly in a microcentifuge to pull bead sample down prior to pelleting with magnetic rack.
?. [Target Protein Binding - Single Tubes]
Place tube in a magnetic rack and remove supernatant. Reserve supernatant as flow through.
?. [Wash - Single Tubes]
Add  to the bead pellet, mix briefly to re-suspend the beads.
[wash buffer]
?. [Wash - Single Tubes]
Place the tube in a magnetic rack to pellet the beads and remove the supernatant.
?. [Elution - Single Tubes]
Add  and mix the suspension for  on a benchtop shaker at 850 rpm.
[elution buffer]
?. [Elution - Single Tubes]
Place the tube in a magnetic rack to pellet the beads, remove and keep the supernatant containing the eluted target protein.
?. [Elution - Single Tubes]
Elution step can be repeated and eluates combined, however the majority of the target protein is in the first elution.
?. [Wash - Single Tubes]
Repeat wash step twice: Add  to the bead pellet, mix briefly to re-suspend the beads.       (1/2)
[wash buffer]
?. [Wash - Single Tubes]
Place the tube in a magnetic rack to pellet the beads and remove the supernatant.                                                       (1/2)
?. [Buffer Preparation for NEBExpress Ni-NTA Magnetic Beads]
To prepare buffers for NEBExpress Ni-NTA Magnetic Beads under Native Conditions: bring all three buffers to a final concentration of pH 7.4To prepare buffers for NEBExpress Ni-NTA Magnetic Beads under Denaturing Conditions: bring all three buffers to a final concentration of or .
[Urea]
[Guanidine]
?. [Bead Equilibration - 96-well plate]
Re-suspend the bead slurry by vortexing or mixing.
?. [Bead Equilibration - 96-well plate]
Immediately dispense  to each well, mixing bead slurry periodically to keep beads evenly suspended.
[bead slurry]
?. [Bead Equilibration - 96-well plate]
Place plate on a 96-Well Microtiter Plate Magnetic Separation Rack and remove the supernatant.
?. [Bead Equilibration - 96-well plate]
Add  to each well and mix briefly.
[binding buffer]
?. [Bead Equilibration - 96-well plate]
Place the plate on the 96-Well Microtiter Plate Magnetic Separation Rack and remove the supernatant.
?. [Target Protein Binding - 96-well plate]
Add up to  to the equilibrated beads.
[crude lysate]
?. [Target Protein Binding - 96-well plate]
Incubate for  at  with a benchtop shaker at 850 rpm.
0 Room temperature
Note: Beads may adhere to sides or cap of the tubes during mixing. Samples can be spun briefly in a microcentifuge to pull bead sample down prior to pelleting with magnetic rack.
?. [Target Protein Binding - 96-well plate]
Place the plate on a 96-Well Microtiter Plate Magnetic Separation Rack and remove supernatant. Reserve supernatant as flow-through sample.
?. [Wash - 96-well plate]
Resuspend the beads in , apply the magnet and remove the supernatant.
[wash buffer]
?. [Wash - 96-well plate]
Repeat wash step twice: Resuspend the beads in , apply the magnet and remove the supernatant.                                                                                                                                                                                   (1/2)
[wash buffer]
?. [Elution - 96-well plate]
Add  and mix the suspension for  on a benchtop shaker at 850 rpm.
[elution buffer]
?. [Elution - 96-well plate]
Apply magnet, remove and keep the supernatant containing the eluted target protein.
?. [Elution - 96-well plate]
Elution step can be repeated and eluates combined, however the majority of the target protein is in the first elution.
?. [Wash - 96-well plate]
Repeat wash step twice: Resuspend the beads in , apply the magnet and remove the supernatant.                                                                                                                                                                                   (2/2)Remove and discard any remaining wash buffer from the bead pellet in the last wash.
[wash buffer]
?. [Wash - Single Tubes]
Repeat wash step twice: Add  to the bead pellet, mix briefly to re-suspend the beads.       (2/2)
[wash buffer]
?. [Wash - Single Tubes]
Place the tube in a magnetic rack to pellet the beads and remove the supernatant.                                                       (2/2)Remove any remaining wash buffer from the bead pellet and discard in the last wash.