Purification using His affinity agarose

 

This protocol applies to all agarose resins 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 Agaroses, featuring NTA, IDA, or the novel EDTA-stable INDIGO ligands. Reagent amounts given apply to 200 mL IPTG-induced bacterial culture of a well-expressed protein (approximately 10–50 mg/L). If other culture volumes are processed or protein expression is higher or lower, reagent volumes may need to be adjusted. 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 based on physical disruption (e.g., sonication or homogenization) or detergents (e.g., CHAPS) can also be used. The His-tagged target protein is purified from the cleared lysate under native conditions in a bind-wash-elute procedure. Binding is performed in batch mode (as opposed to on-column binding). This method is most efficient, especially when the target protein is present at low concentrations or the His-tag is not fully accessible.

Buffer compositions - Native protein purification protocol

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.

Wash Buffer, 100 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 6 mL
Imidazole 20 mM 68.08 1 M 6.8 g/ 100 mL 2 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.

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 5 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.

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 pellets corresponding to 200 mL bacterial culture on ice for 15 min. 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 10 mL Native Lysis Buffer supplemented with 1 mg/mL lysozyme, and pour it into a 50 mL conical centrifuge tube.
  3. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption.
  4. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4 °C for 1 h, depending on the temperature stability of the protein.
  5. Centrifuge the lysate for 30 min at 10,000 x g and 2-8 °C. Carefully collect the supernatant without touching the pellet. 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 Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50 % suspension (corresponding to 500 μL bed volume) to a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column.
  7. Add 2.5 mL Native Lysis Buffer and gently resuspend the slurry to equilibrate the resin. Allow the resin to settle by gravity and remove 2 mL supernatant.
  8. Add 10 mL cleared lysate to the equilibrated PureCube Ni-NTA Agarose resin and incubate at 4 °C for 1 h on an end-over-end shaker. Tip: Alternatively, batch binding can be performed directly in a gravity flow column with closed bottom and top outlets.
  9. Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use Lysis Buffer to rinse the centrifuge tube and remove resin adhered to the wall.
  10. Remove the bottom cap of the column and collect the flow-through.
  11. Wash the column with 5 mL Native Wash Buffer. Repeat the washing step at least 3 times.
  12. Elute the His-tagged protein 5 times using 0.5 mL Native Elution Buffer. Collect each eluate in a separate tube and determine the protein concentration of each fraction. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  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.

 

Buffer compositions - Denaturing protein purification protocol

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, 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 6.3 with HCl and add water to a total volume of 50 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.
   
 B.Protocol for purification under denaturing conditions:
 
 
  1. Thaw the E. coli cell pellet on ice.
  2. Resuspend the cell pellet in 10 mL Denaturing Lysis Buffer. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). Nucleic acids can also 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. Resuspend the PureCube Ni-NTA Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension (corresponding to 0.5 mL bed volume) into a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant.
  6. Add the cleared lysate to the resin and incubate the mixture for 1 h at room temperature on an end-over-end shaker. Tip: Alternatively, batch binding can be done directly in a gravity flow column with closed top and bottom outlet.
  7. Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use Lysis Buffer to rinse the centrifuge tube and remove resin adhered to the wall.
  8. Remove the bottom cap of the column and collect the flow-through.
  9. Wash the column with 5 mL Denaturing Wash Buffer. Repeat the washing step at least 3 times.
  10. Elute the His-tagged protein 5 times using 0.5 mL Denaturing Elution Buffer. Collect each eluate in a separate tube and determine the protein concentration of each fraction. Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  11. 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.
  12. Optional: Perform Western Blot experiment using PentaHis Antibody.
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