cOmplete™ His-Tag Purification Column Protocol & Troubleshooting

Purification Protocols

cOmplete His-Tag Purification Columns (Product No. COHISC-RO) are compatible with automated chromatography systems such as ÄKTAexplorer.

Purification Under Native Conditions

Purification of native proteins should be performed using optimal buffer conditions for the target protein. Buffers recommended in this document are well established examples and can be adapted to achieve optimal conditions for a specific target protein.
cOmplete His-Tag Purification Columns offer flexibility in selecting the optimal buffer conditions.
Warning: The binding capacity may drop significantly if the buffer composition is suboptimal.
Note: For best results, load with a low flow rate to bind the target proteins more efficiently to the resin.
Warning: cOmplete His-Tag Purification Columns have been optimized using Buffer A and Buffer B specified in the table below. Other buffers might function as well, but need to be tested before use with cOmplete His-Tag Purification Columns.
Buffer A: 50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl
Buffer B: 50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 250 mM imidazole
Note: The following protocol describes the experimental procedures when using an ÄKTAexplorer 100 System (Cytiva) for FPLC purification.

1. Wash the pump with 10 to 20 mL Buffer A using the System Wash function of the ÄKTAexplorer System at a flow rate of 10 mL/min. Ensure that all air is displaced from the pumps and tubings of the system.
2. Remove the plug at the column outlet and attach it to the outlet tubing of the ÄKTAexplorer System. Note: Save the plug of the column outlet in case the column needs to be stored or is planned to be reused.
3. As soon as Buffer A is running out of the inlet tubing of the ÄKTAexplorer System, remove the upper plug from the column and immediately attach it to the inlet tubing of the ÄKTAexplorer System. Continuously measure OD₂₈₀ values on the ÄKTAexplorer System. Note: If a fluorescent protein is purified, continuously measure the OD values at the absorption maximum of the fluorescence dye; e.g., OD₄₈₅ for GFP (Green Fluorescent Protein) or OD₄₃₅ for CFP (Cyan Fluorescent Protein). Note: Save the plug of the column inlet in case the column needs to be stored or is planned to be reused.
4. Define the flow rate as 10 mL/min for the 5 mL column or 2 mL/ min for the 1 mL column and equilibrate the column with 10 column volumes of Buffer A.
5. Pause the run. Load the cleared sample (e.g., after an ultracentrifugation or filtration step) onto the column with a volumetric flow rate of 2.5 mL/min for the 5 mL column or 0.5 to 1 mL/min for the 1 mL column. Warning: To prevent blockage of the column, remove insoluble material before loading the column. Warning: Since the binding specificity of the resin is high, the kinetics of adhesion of the protein to the resin is slower than other available resins. If using high volumetric flow rates for loading, protein yield can decrease.
6. Wash the column with Buffer A until the OD₂₈₀ value reaches the baseline level (approximately 10 column volumes).
7. Elute the His-tagged protein with a gradient of Buffer A (without imidazole) and Buffer B (250 mM imidazole). Warning: Protein peaks can be expected between 25 to 45 mM imidazole. Due to the specific characteristics of cOmplete His-Tag Purification Columns, a protein can already be eluted with approximately 25 mM imidazole. Warning: The amount of imidazole required in the elution buffer for efficient release of the target protein from the resin depends on various parameters, such as: • the length of the His-tag, • the accessibility of the His-tag.
8. Wash and equilibrate for the next run. For details, refer to section Cleaning Procedures. Warning: If the column is not immediately reused, clean the column with 2 column volumes of 2 M imidazole to remove nonspecific binding of proteins. Equilibrate the column in a 20% ethanol solution and tightly close the column at both threads with plugs. Store at +2 to +8 °C to prevent cell growth.

Note: Refer to sections Purification Process Optimization and Troubleshooting for technical advice in optimizing the purification results.

Purification Under Denaturing Conditions

Purification of denatured proteins should be performed using optimal buffer conditions for the target protein. Buffers recommended in this document are well established examples and can be adapted to achieve optimal conditions for a specific target protein.
cOmplete His-Tag Purification Columns offer flexibility in selecting optimal buffer conditions.
Denature the protein or dissolve the inclusion bodies in a buffer containing 6 M guanidinium-HCl or 8 M urea.
Warning: The addition of urea to buffered solutions will cause the pH to drop. It is essential to adjust the pH of the buffer with NaOH after urea addition.
Warning: The binding capacity may also drop significantly if the buffer composition is suboptimal.
Note: For best results, load with a low flow rate to bind the target proteins more efficiently to the resin.
Warning: cOmplete His-Tag Purification Columns have been optimized using Buffer C, Buffer D, Buffer E, and Buffer F specified in the table below. Other buffers might function as well, but need to be tested before use with cOmplete His-Tag Purification Columns.
Buffer C: 100 mM NaH₂PO₄; 10 mM Tris-HCl; 8 M urea; pH 8.0
Buffer D: 100 mM NaH₂PO₄; 10 mM Tris-HCl; 8 M urea; pH 6.3
Buffer E: 100 mM NaH₂PO₄; 10 mM Tris-HCl; 8 M urea; pH 5.9
Buffer F: 100 mM NaH₂PO₄; 10 mM Tris-HCl; 8 M urea; pH 4.5
Note: The following protocol describes the experimental procedures when using an ÄKTAexplorer 100 System (Cytiva) for FPLC purification.

1. Wash the pump with 10 to 20 mL Buffer C using the System Wash function of the ÄKTAexplorer System at a flow rate of , e.g., 10 mL/min. Ensure that all air is displaced from the pumps and tubings of the system.
2. Remove the plug at the column outlet and attach it to the outlet tubing of the ÄKTAexplorer System. Note: Save the plug of the column outlet in case the column needs to be stored or is to be reused.
3. As soon as Buffer C is running out of the inlet tubing of the ÄKTAexplorer System, remove the upper plug from the column and immediately attach it to the inlet tubing of the ÄKTAexplorer System. Continously measure OD₂₈₀ values on the ÄKTAexplorer System. Note: If a fluorescent protein is purified, continously measure the OD values at the absorption maximum of the fluorescence dye; e.g., OD₄₈₅ for GFP (Green Fluorescent Protein or OD₄₃₅ for CFP (Cyan Fluorescent Protein). Note: Save the plug of the column inlet in case the column needs to be stored or is to be reused.
4. Define the flow rate as 10 mL/min for the 5 mL column or 2 mL/min for the 1 mL column and equilibrate the column with 10 column volumes of Buffer C.
5. Pause the run. Load the cleared sample (e.g., after an ultracentrifugation or filtration step) onto the column with a volumetric flow rate of 2.5 mL/min for the 5 mL column or 0.5 to 1 mL/min for the 1 mL column. Warning: To prevent blockage of the column, remove insoluble material before loading the column. Warning: Since the binding specificity of the resin is high, the kinetics of adhesion of the protein to the resin is slower than other available resins. If using high volumetric flow rates for loading, protein yield can decrease.
6. Wash the column with Buffer C until the OD₂₈₀ value reaches the baseline level (approximately 10 column volumes).
7. Wash with 10 to 20 column volumes of Buffer D.
8. Wash with 10 to 20 column volumes of Buffer E.
9. Elute the His-tagged protein with 10 to 20 column volumes of Buffer F. Note: Alternatively, the elution can also be performed with a gradient up to 200 mM imidazole solution using Buffer A and Buffer B instead of the pH shift option (refer to the elution step within section Purification Under Native Conditions).
10. Wash and equilibrate for the next run under denaturing conditions with Buffer C or wash with Buffer A to remove the denaturing agents if the column will next be used under native conditions. For details, refer to section Cleaning Procedures. Warning: If the column is not immediately reused, clean the column with 2 column volumes of 2 M imidazole to remove nonspecific binding of proteins. Equilibrate the column in a 20% ethanol solution and tightly close the column at both threads with plugs. Store at +2 to +8 °C to prevent cell growth.

Note: Refer to section Purification Process Optimization and Troubleshooting for technical advice in optimizing the purification results.

Cleaning Procedures

cOmplete His-Tag Purification Columns can be used multiple times without loss of binding capacity. Over time, however, some protein aggregates might accumulate, leading to a decrease of efficiency of the resin within the columns. This can be identified by a slower flow rate or a higher back pressure.
The cleaning procedures remove aggregates for further efficient use of the columns. Different cleaning procedures can be carried out, based on the different applications. Once the cleaning procedure is completed, the resin should be transferred to 20% ethanol.

Stringent Native Cleaning

This method is appropriate when non-aggregating proteins have been purified, and if the column is used again for purifying the same protein.

• Wash with 10 column volumes of 1 M imidazole/HCl, pH 7.5,
• Wash with 10 column volumes of 4 M imidazole/HCl, pH 7.5,
• Equilibrate the column with binding buffer and proceed to the next round of purification or transfer the material to 20% ethanol.

Denaturing Cleaning with SDS

This method is appropriate to remove aggregated proteins and lipids.
Warning: This cleaning procedure has to be performed at +15 to +25 °C because the solubility of SDS is more effective at this temperature than at +2 to +8 °C.
Note: The SDS buffer may also contain 50 mM DTT.
Warning: Avoid using K⁺ in this buffer to prevent precipitation with SDS.

• Wash with 10 column volumes of 1 M imidazole/HCl, pH 7.5,
• Wash 2 times with 10 column volumes of 1 M imidazole/HCl, pH 7.5, 20% ethanol, 2 to 4% SDS,
• Remove SDS with 3 times 10 column volumes of 20% ethanol.

Denaturing Cleaning with Guanidinium-HCl

This method is appropriate to remove aggregated proteins.
Note: The guanidinium-HCl buffer may also contain 50 mM DTT.

• Wash with 10 column volumes of 1 M imidazole/HCl, pH 7.5,
• Wash twice with 10 column volumes of 6 M guanidinium-HCl, 1 M imidazole, pH7.5,
• Wash twice with 10 column volumes of 20% ethanol.

Note: In general, the choice of cleaning method depends on the protein type.
Note: The denaturing cleaning procedure with guanidinium-HCl presents fewer constraints than the denaturing cleaning method with SDS.

Purification Process Optimization

The parameters allowing for the maximal protein yield and purity might vary significantly depending on the characteristics of a given target protein. To optimize the protein purification procedure for highest protein purity, determine the optimal operating conditions for the specific target protein. Both purity and yield of a protein preparation depends on the sample amount. If the amount of sample is too high, the resin's binding capacity may not be sufficient to bind all target protein, and this will result in a suboptimal protein yield. If the amount of sample is too low, the remaining binding sites on the resin may enable background binding of lysate components. Optimal results are obtained when the amount of target protein matches the amount of resin within the columns. The capacity for a given target protein depends on several factors such as target protein size, conformation, multimerization status, length and accessibility of the His-tag, expression level and solubility of the His-tagged protein, lysate concentration, as well as the buffer pH and composition. For best results, determine the optimal ratio of the volume of lysate and resin within the columns required for the purification of a specific protein of interest, which is dependent on the expression rate of the protein:

• Incubate the columns with varying volumes of lysates, in parallel test experiments,
• Wash the columns and elute the bound proteins,
• Determine the amount of target protein in the unbound fractions and in the eluate by SDS-PAGE,
• The volume of lysate is optimal when only a small amount of target protein remains in the flow through and the maximal amount of protein is detected in the eluate fractions.

Note: The yield of the target protein can be optimized by allowing more time for the protein to bind to the resin. This can be performed by reducing the flow rate during the loading step of the chromatography purification.
Note: The optimal concentration of imidazole during binding, washing and elution steps can also be determined during pretrial experiments.
Note: Optimal results can typically be achieved with buffers containing a high salt concentration (300 mM) at pH 8.0 for target proteins compatible with those conditions.

Troubleshooting

Problem; Possible Cause; Recommendation

Bubbles form in the bed resin:

• Mixing of the storage buffer (20% ethanol) with aqueous buffer; a) After storage at +2 to +8 °C, equilibrate the columns to +15 to +25 °C. b) Degas the buffer before equilibration of the column.

The sample does not flow easily through the columns (low flow rate or high back pressure):

•  Particulates from the lysates may have clogged the columns; a) Centrifuge or ultracentrifuge the sample before loading on the column. b) Reduce the flow rate. c) Clean the columns using a denaturing cleaning procedure.

Inefficient binding of the target protein to the resin within the columns:

• Suboptimal buffer conditions during the binding step; Lower the imidazole concentration and/or increase the pH during the binding step.
• Incubation time is too short; a) Extend the incubation time. b) Lower the flow rate during binding.
• The His-tag is not accessible; a) Change the position of the His-tag. b) Use a longer His-tag.

Inefficient or no elution of the target protein:

• The target protein multimerizes and binds more avidly to the resin; Increase the imidazole concentration during elution.
• The protein precipitates on the resin before elution; a) Increase ionic strength to minimize isoelectric precipitations. b) Elute under denaturing conditions.
• The target protein precipitates during a pH shift elution; Elute with imidazole instead.
  

Recovery of the target protein is too low:

• The target protein may be degraded; a) Add protease inhibitors to the sample if degradation occurs during cell lysis. b) Protein degradation can also be prevented by working at +2 to +8 °C.
  
• The His-tag might not be accessible; a) Use a longer His-tag. b) Check if the target protein contains the His-tag. c) Optimize expression conditions and buffers. d) Change the localization of the His-tag.
• The His-tag might have been digested by proteases; a) Change to another expression host. b) Use protease inhibitors.
• The target protein might not be soluble; a) Lower the expression temperature, strength, and duration of induction. b) Purification under denaturing conditions. c) Include solubility-enhancing fusion partners.
• The resin is limiting; Verify that the expressed His-tagged protein is proportionate to the resin within the columns.

Target protein elutes with contaminants:

• The host proteins interact with the resin; a) Increase the stringency during the loading and washing step by increasing the imidazole concentration/lowering the pH. b) Increase the amount of the sample. c) Wash the column with a stringent buffer.
• DNA and/or RNA contaminants; a) Purify under denaturing conditions. b) Include a DNase I digestion step and/or a Polymin P-mediated precipitation step before adding the lysate to the columns.

Target protein is degraded during or following the cell lysis:

• Insufficient protection from proteases; a) Add protease inhibitors to the buffers and/or culture. b) Optimize the experimental workflow. c) Strictly work on ice.
  

Target protein is degraded in the host cell:

• a) Use a protease-deficient host strain. b)Reduce the induction time.