cOmplete™ His-Tag Purification Resin Protocol & Troubleshooting

Protocol for His-Tag Resin

Product No. COHISR-RO

FPLC Purification Under Native Conditions Using the ÄKTAexplorer 100 System

A detailed procedure for automated Fast Protein Liquid Chromatography (FPLC) using the ÄKTAexplorer 100 System is available in the Instructions for Use of cOmplete His-Tag Purification Column (06781535001, 06781543001), see chapter Purification Protocols > Purification Under Native Conditions.

FPLC Purification Under Denaturing Conditions Using the ÄKTAexplorer 100 System

A detailed procedure for automated Fast Protein Liquid Chromatography (FPLC) using the ÄKTAexplorer 100 System is available in the Instructions for Use of cOmplete His-Tag Purification Column (06781535001, 06781543001), see chapter Purification Protocols > Purification Under Denaturing Conditions.

Chromatography Purification Protocols

Purifications with cOmplete His-Tag Purification Resin are compatible with various general purification formats including batch processing, column purification with gravity flow and automated processing using an automated chromatography system.

Column Packing Procedure for FPLC Applications

cOmplete His-Tag Purification Resin is supplied as a 50% suspension in 20% ethanol.

1. Resuspend cOmplete His-Tag Purification Resin by inverting the bottle several times.
2. Transfer the appropriate amount of resin to a chromatography column with a pipette, ensuring that no air bubbles get trapped in the adapter or gel bed.
3. Allow for the resin to settle.
4. Let the excess buffer drain through the column by gravity flow.
5. Insert the top adapter and adjust it to the top of the bed.
6. Connect the column to a chromatography system/buffer reservoir.
7. Use a flow rate as described in section Technical Specifications.

Purification Under Native Conditions

Purification of native protein 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 Resin offers full flexibility in selecting the optimal buffer conditions without compromises.
Warning: cOmplete His-Tag Purification Resin has been optimized with the following Buffer A and Buffer B specified below. Other buffers might function as well, but need to be tested prior to use with cOmplete His-Tag Purification Resin.
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

1. Equilibrate the column with 10 column volumes of Buffer A.
2. Load the cleared sample containing the His-tagged protein (e.g., after an ultracentrifugation or filtration step) onto the column with a volumentric flow rate of 2.5 mL/min for 5 mL bed volume of resin or 0.5 to 1 mL/min for 1mL bed volume. Warning: To prevent blockage of the resin, remove insoluble material prior to 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.
3. Wash the column with Buffer A until the absorption (A₂₈₀) reaches the baseline level (approximately 10 column volumes).
4. 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 Resin, 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: the length of the His-tag, the accessibility of the His-tag.
5. Wash and equilibrate for the next run (refer to section Cleaning Procedure). Warning: If the purification 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 store at 2-8 °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

Buffers recommended in this document are well established examples and can be adapted to achieve optimal conditions for a specific target protein.
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, perform an overnight incubation to bind denatured target proteins more efficiently to the resin.
Warning: cOmplete His-Tag Purification Resin has been tested with the following buffers and therefore functions optimally with these buffers. Other buffers might function and need to be tested prior to use with cOmplete His-Tag Purification Resin.
Prepare the following buffers for the chromatography procedure under denaturing conditions:
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

1. Equilibrate the column with 10 column volumes of Buffer C.
2. Load the cleared sample containing the His-tagged protein (e.g., after an ultracentrifugation or filtration step) onto the column with a flow rate of 0.5-1 mL/min for 1 mL bed volume of resin. Warning: To prevent blockage of the resin, 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. For this reason, do not exceed a 1 mL/min flow rate during the protein loading step. If using high volumetric flow rates for loading, protein yield can decrease.
3. Wash the column with Buffer C until the absorption (A280) reaches the baseline level (approximately 10 to 20 column volumes).
4. Wash with 10 to 20 column volumes of Buffer D.
5. Wash with 10 to 20 column volumes of Buffer E.
6. Elute with 10 to 20 column volumes of Buffer F.
7. 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. Warning: If the column is not immediately reused, clean the column with two column volumes of 2 M imidazole. Equilibrate the column in a 20% ethanol solution, and store at 2-8 °C to prevent cell growth. Note: Alternatively, the elution can also be performed with a gradient up to 250 mM imidazole solution instead of the pH shift option.

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

Batch Purification Protocol
The batch purification protocol can be conducted under native conditsions as well as under denaturing conditions.

1. Transfer the appropriate amount of cOmplete His-Tag Purification Resin to a graduated container or an empty column.
2. Equilibrate the column with the resin with approximately 20 column volumes of Buffer A.
3. Add an appropriate amount of cleared lysate to the prepared resin slurry. Gently swirl the mixture and incubate the resin-lysate mixture at 2-8 °C for 2-12 hours on a shaker.
4. Load the resin-lysate mixture onto an appropriate sized column.
5. Collect the column flow through.
6. Wash the column with at least 5 column volumes of Buffer A.
7. Elute the protein with at least 5 column volumes of Buffer B.
8. Wash and equilibrate with Buffer A for the next run. Warning: If the column is not immediately reused, equilibrate the column in a 20% ethanol solution, and store at 2-8 °C to prevent cell growth.

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

Cleaning Procedures

cOmplete His-Tag Purification Resin 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. This can be identified by a slower flow rate or a higher back pressure. The cleaing procedures remove aggregates for further efficient use of the resin. Different cleaning procedures can be carried out, based on the different applications of the resin. Once the cleaning procedure is completed, the resin should be transferred 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 must be performed at 15-25 °C because the solubility of SDS is more effective at this temperature than at 2 -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 2 times with 10 column volumes of 6 M guanidinium-HCl, 1 M imidazole, pH 7.5,
• Wash 2 times 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. For best results, optimize the key parameters in small-scale trial purifications. To optimize the protein purification procedure for highest protein purity, determine the optimal operating conditions of the resin for the specific target protein. Both purity and yield of a protein preparation depends on the amount of cOmplete His-Tag Purification Resin used for binding. If the amount of resin is too high in relation to the amount of available target protein, the remaining binding sites on the resin may enable background binding of lysate components. If the amount of resin is too low, the resin´s binding capacity may not be sufficient to bind all target protein, and this will result in a suboptimal protein yield. Optimal results are obtained when the capacity of the matrix matches the amount of target protein. The capacity for a given target protein depends on several factors such as target protein size, conformation, and 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 volume of resin required for the purification of a specific protein of interest. Based on the expression rate of the protein and the volume of lysate available, determine the optimal volume of resin by performing the following pretrial:

• Incubate a small quantity of settled resin with varying volumes of lysates, in parallel test experiments,
• Wash the resin 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/volume of resin ratio 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.

For example, for the purification of 20 to 40 mg T7 RNA polymerase (97 kDa), the amount of resin needed is 1 mL.
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. Alternatively, batch adsorption can be carried out for up to 12 hours during a batch purification procedure, without impacting the protein integrity. For example, the adsorption of the His6-tagged T4 gene 32 protein has been measured during a batch purification procedure after different incubation times of the protein solution with cOmplete His-Tag Purification Resin in a tube, on a roller.
Note: The optimal concentration of imidazole during binding, washing and elution steps can also be determined during pretrial experiments with a fixed aliquot of resin. This identifies the optimal amount of lysate for saturating that aliquot of resin using varying concentrations of imidazole.
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 2-8 °C, equilibrate the columns to 15-25 °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 2-8 °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.