Ni-NTA MagBeads

• High binding capacity >70 mg/mL settled beads
• Stable in buffer containing 10 mM DTT and 1 mM EDTA
• Delivered as 25 % suspension
• Average magnetic agarose bead size: 25 μm

• Lysis Buffer
• Wash Buffer
• Elution Buffer
• Ice bath
• Refrigerated microcentrifuge (min 10,000 x g)
• Micropipettor and Micropipetting tips
• 1.5 mL conical microcentrifuge tubes
• Magnetic holder for microcentrifuge tubes (for separation of magnetic beads)
•  pH meter
• End-over-end shaker
• SDS-PAGE buffers, reagents and equipment
  Optional: Western Blot reagents and equipment

KD of NTA to 6xHis-tag: ca 10 μM

Sample Volume for an assay: >10 mL E.coli culture volume or corresponding quantity. Protocol can be scaled up easily.

A Purification under native conditions:

• Native Lysis buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 10 mM, pH 8
  Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer.
  Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1-5 mM.
• Native Wash buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 20 mM, pH 8
• Native Elution buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 250- 500 mM, pH 8
  
  Additional chemicals required: Lysozyme, Benzonase® nuclease,
  Optional: Protease inhibitor cocktail

• Denaturing Lysis buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M, pH 8.0,
  Optional: Benzonase® nuclease (e.g. Merck Milipore, #707464)
• Denaturing Wash buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M, pH 6.3NaH2PO4 100 mM
• Denaturing Elution buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M, pH 4.5
  Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  Note: Due to urea dissociation, adjust the pH immediately before use.

A Protocol for purification under native conditions:

1. Thaw the E. coli cell pellet on ice. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
2. Resuspend the cell pellet in 1 mL Native Lysis Buffer supplemented with 1 mg/mL lysozyme.
3. Add 6 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
4. Incubate for 30 min on ice, if necessary. Otherwise, incubating at room temperature (20-25 °C) may be more efficient.
5. Centrifuge the lysate for 30 min at 10,000xg and 4 °C. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
6. Resuspend the HighSpec Ni-NTA MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic bead suspension into a conical microcentrifuge tube. Note: Depending on the protein expression rate, the quantity of magnetic bead suspension can be adjusted from 2-200 μL.
7. Add 500 μL Native Lysis Buffer and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant.
8. Pipet 1 mL of the cleared lysate onto the equilibrated magnetic beads, and incubate the lysate-magnetic bead mixture at 4 °C for 1 h on an end-over-end shaker.
9. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
10. Remove the tube from the magnet. Add 500 μL Native Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
11. Repeat step 10 twice.
12. Elute the His-tagged protein using 100 μL Native Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
13. Repeat step 12xxx Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
14. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate the sample at 46 °C for 30 min in preparation for SDS-PAGE analysis.
15. Optional: Perform Western Blot experiment using PentaHis Antibody.

B Protocol for purification under denaturing conditions:

1. Thaw the E. coli cell pellet on ice.
2. Resuspend the cell pellet in 1 mL Denaturing Lysis Buffer. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). In addition, nucleic acids can be sheared by passing the lysate 10 times through a fine-gauge needle.
3. Incubate at room temperature for 30 min on an end-over-end shaker.
4. Centrifuge the lysate for 30 min at room temperature and 10,000 x g. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
5. Pipet 1 mL of the cleared lysate into a conical microcentrifuge tube.
6. Resuspend the HighSpec Ni-NTA MagBeads by vortexing. Transfer 40 μL of the 25% magnetic beads suspension onto the lysate. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 µl.
7. Incubate the lysate-magnetic bead mixture at room temperature for 1 h on an end-over-end shaker.
8. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Tip: Briefly centrifuge the sample before placing it on the magnetic separator in order to collect liquid from the lid.
9. Remove the tube from the magnet. Add 500 µL Denaturing Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
10. Repeat step 9 twice.
11. Elute the His-tagged protein using 100 μL Denaturing Elution Buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 µl.
12. Repeat step 11. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
13. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46˚C for 30 min in preparation for SDS-PAGE analysis.
14. Optional: Perform Western Blot experiment using PentaHis Antibody.Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.

Analyze by SDS-PAGE, Bradford Assay or spectrophotometrically.