Home Protocols NEBExpress® Ni-NTA Magnetic Beads Typical Reaction Protocol

NEBExpress® Ni-NTA Magnetic Beads Typical Reaction Protocol

NEBExpress® Ni-NTA Magnetic Beads can be used for the purification of His-tagged fusion proteins under native or denaturing conditions

  • 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.

  • For optimal performance, the amount of beads used should match the approximate amount of His-tagged protein to be captured.

  • 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.

  • 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)

Buffer Preparation for NEBExpress Ni-NTA Magnetic Beads:

Supplied Concentrated Buffers:

B1076, 2X IMAC Buffer (0.04 M Sodium Phosphate, 0.6 M NaCl, pH 7.4)
B1077, 2M Imidazole (2M Imidazole, pH 7.4)

To prepare buffers for NEBExpress Ni-NTA Magnetic Beads under Native Conditions


 Lysis/Binding Buffer:
20 mM sodium phosphate,
300 mM NaCl,
10 mM Imidazole,
pH 7.4
 Wash Buffer:
20 mM sodium phosphate,
300 mM NaCl,
20 mM Imidazole,
pH 7.4 		 Elution Buffer:
20 mM sodium phosphate,
300 mM NaCl,
500 mM Imidazole,
pH 7.4 
2X IMAC Buffer 1.5 ml
 10 ml
 1.25 ml
2M Imidazole 
 0.015 ml 
 0.2 ml 
 0.625 ml
H2O   1.49 ml 
 9.8 ml  
 0.625 ml
Total 
 3.0 ml 
 20 ml 
 2.5 ml

 

Preparation of NEBExpress Ni-NTA Magnetic Beads under Denaturing Conditions:

  1. Prepare Lysis/Binding, Wash and Elution Buffers as described in the table above

  2. Bring all three buffers (Lysis/Binding, Wash and Elution Buffers) to a final concentration of 8M Urea or 6M Guanidine.

Notes:

  1. Working buffers are sufficient for 10 reactions using the supplied concentrated buffers; volumes can be scaled up for larger reactions

  2. 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.

Protocol using Single Tubes

Bead Equilibration

  1. Resuspend the bead slurry by vortexing or mixing.

  2. Immediately dispense 50 µl of the bead slurry to a 1.5 ml tube.

  3. Add 200 µl of binding buffer to the bead slurry and mix briefly.

  4. Place the tube in a magnetic rack to pellet the beads, remove and discard the supernatant.

Target Protein Binding

  1. Add 1.0 ml of the crude lysate to the equilibrated beads.

  2. Incubate for 30 min at room temperature (RT) with end over end mixing or with a benchtop shaker at 850 rpm.

  3. 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.

  4. Place tube in a magnetic rack and remove supernatant. Reserve supernatant as flow through.

Wash

  1. Add 500 µl of wash buffer to the bead pellet, mix briefly to re-suspend the beads.

  2. Place the tube in a magnetic rack to pellet the beads and remove the supernatant. Repeat wash step twice. Remove any remaining wash buffer from the bead pellet and discard in the last wash. 

Elution

  1. Add 100 µl of elution buffer and mix the suspension for 2 minutes on a benchtop shaker at 850 rpm.

  2. Place the tube in a magnetic rack to pellet the beads, remove and keep the supernatant containing the eluted target protein.

  3. Elution step can be repeated and eluates combined, however the majority of the target protein is in the first elution.

Protocol using 96-well plates

Bead Equilibration

  1. Re-suspend the bead slurry by vortexing or mixing.

  2. Immediately dispense 50 µl of the bead slurry to each well, mixing bead slurry periodically to keep beads evenly suspended.

  3. Place plate on a 96-Well Microtiter Plate Magnetic Separation Rack and remove the supernatant.

  4. Add 200 µl of binding buffer to each well and mix briefly.

  5. Place the plate on the 96-Well Microtiter Plate Magnetic Separation Rack and remove the supernatant.

Target Protein Binding

  1. Add up to 200 µl of the crude lysate to the equilibrated beads.

  2. Incubate for 30 min at RT with a benchtop shaker at 850 rpm.

    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.

  3. Place the plate on a 96-Well Microtiter Plate Magnetic Separation Rack and remove supernatant. Reserve supernatant as flow-through sample.

Wash

  1. Resuspend the beads in 200 µl of wash buffer, apply the magnet and remove the supernatant.

  2. Repeat wash step twice. Remove and discard any remaining wash buffer from the bead pellet in the last wash.

Elution

  1. Add 100 µl of elution buffer and mix the suspension for 2 minutes on a benchtop shaker at 850 rpm.

  2. Apply magnet, remove and keep the supernatant containing the eluted target protein.

  3. Elution step can be repeated and eluates combined, however the majority of the target protein is in the first elution.