Purification of His-tagged Proteins Under Native/Denaturing Conditions Using PureCube His Affinity MagBeads

This protocol applies to all magnetic beads that are listed as His affinity Products in our online shop!
This protocol describes the generation of a cleared lysate from an E. coli cell pellet and the subsequent purification of His-tagged proteins under native conditions using our PureCube His Affinity MagBeads, featuring NTA, IDA, or the novel EDTA-stable INDIGO ligands. Reagent amounts given apply to 10 mL IPTG-induced bacterial culture of a well-expressed protein (approximately 10–50 mg/L). Magnetic bead purification is easily scalable. To minimize unspecific binding and reduce cost, the volume magnetic bead suspension used should be adjusted to the expression level of interest. See Table 1 for more details. In this protocol, cell lysis is done using lysozyme because it is an inexpensive and efficient method for cells that have been frozen. However, lysis methods using detergents (e.g., CHAPS) can also be used. The His-tagged target protein is purified from cleared lysate under native conditions in a bind-wash-elute procedure.

Buffer compositions - His Tag purification native and denaturing

Native Lysis Buffer, 50 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
NaH2PO4 50 mM 119.98 0.5 M 29.99 g/ 500 mL 5 mL
NaCl 300 mM 58.44 5 M 146.1 g/ 500 mL 3 mL
Imidazole 10 mM 68.08 1 M 6.8 g/ 100 mL 0.5 mL
Instructions: Mix in 40 mL water. Adjust the pH to 8.0 using NaOH and then add water to a total volume of 50 mL. Always prepare fresh.

Native Wash Buffer, 50 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
NaH2PO4 50 mM 119.98 0.5 M 29.99 g/ 500 mL 5 mL
NaCl 300 mM 58.44 5 M 146.1 g/ 500 mL 3 mL
Imidazole 20 mM 68.08 1 M 6.8 g/ 100 mL 1 mL
Instructions: Mix in 80 mL water. Adjust the pH to 8.0 using NaOH and then add water to a total volume of 100 mL. Always prepare fresh.

Native Elution Buffer, 50 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
NaH2PO4 50 mM 119.98 0.5 M 29.99 g/ 500 mL 10 mL
NaCl 300 mM 58.44 5 M 146.1 g/ 500 mL 3 mL
Imidazole* 500 mM 68.08 1 M 6.8 g/ 100 mL 25 mL
Instructions: Mix in 40 mL water. Adjust the pH to 8.0 using NaOH and then add water to a total volume of 50 mL. Always prepare fresh.
*Tag length and protein structure can impact the interaction between His-tag and nickel ion. Therefore, we recommend trying a concentration gradient of imidazole to find the minimum concentration that elutes the desired amount of protein from the column.

Denaturing Lysis Buffer, pH 8.0, 50 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
NaH2PO4 100 mM 119.98 0.5 M 29.99 g/ 500 mL 10 mL
Tris base 10 mM 121.14 1 M 12.11 g/ 100 mL 0.5 mL
Urea 8 M 60.06 - - 24 g
Instructions: Dissolve urea in 30 mL water and then add the remaining components. Adjust pH to 8.0 with HCl and add water to a total volume of 50 mL. Due to urea dissociation, adjust the pH immediately before use.

Denaturing Wash Buffer, pH 6.3, 100 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
NaH2PO4 100 mM 119.98 0.5 M 29.99 g/ 500 mL 20 mL
Tris base 10 mM 121.14 1 M 12.11 g/ 100 mL 1 mL
Urea 8 M 60.06 - - 48 g
Instructions: Dissolve urea in 60 mL water, then add remaining components. Adjust pH to 6.3 with HCl and add water to a total volume of 100 mL. Due to urea dissociation, adjust the pH immediately before use.

Denaturing Elution Buffer, pH 4.5, 50 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
NaH2PO4 100 mM 119.98 0.5 M 29.99 g/ 500 mL 10 mL
Tris base 10 mM 121.14 1 M 12.11 g/ 100 mL 0.5 mL
Urea 8 M 60.06 - - 24 g
Instructions: Dissolve urea in 30 mL water, then add remaining components. Adjust pH to 4.5 with HCl and add water to a total volume of 50 mL. Due to urea dissociation, adjust the pH immediately before use.

5X SDS Page Buffer, 10 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
Tris-HCl, pH 6.8–7.0 300 mM 121,14 1 M 121,14 g/ 1 L 3 mL
Glycerol 50% (v/v) - 100% (v/v) - 5 mL
SDS 5% (w/v)) - - - 0.5 g
Bromophenol blue 0.05% (w/v) - 4% - 125 μL
DTT 250 mM 154.25 1 M 1.54 g/ 10 mL 125 μL/aliquot
Instructions: Make sure to prepare a 1 M Tris-HCl stock by dissolving Tris base in 500 mL deionized water, adding HCl to a pH of 6.8–7.0, and adding water to a final volume of 1 L. For the SDS-PAGE Buffer, mix all components listed except DTT and add water to a total of 10 mL. Freeze 20 aliquots (375 µL each) at –20˚C. Before use, add DTT to the needed single aliquots.

 

   
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 PureCube 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 12 three times. 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.
This protocol describes the generation of a cleared lysate from an E. coli cell pellet and the subsequent purification of His-tagged proteins under denaturing conditions our PureCube His Affinity MagBeads, featuring NTA, IDA, or the novel EDTA-stable INDIGO ligands. Reagent amounts given apply to 10 mL IPTG-induced bacterial culture of a well-expressed protein (approximately 10–50 mg/L). Magnetic bead purification is easily scalable. To minimize unspecific binding and reduce cost, the volume magnetic bead suspension used should be adjusted to the expression level of interest. See Table 1 for more details. In this protocol, cells are lysed with a high concentration of urea, which also aids to dissolve insoluble protein aggregates. The His-tagged protein is purified from the cleared lysate under denaturing conditions in a bindwash-elute procedure. Binding occurs at slightly alkaline pH, while washing and elution are done with a stepwise pH decrease.

 

   
 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 PureCube 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. If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
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