protocol

Introduction

The Ni-NTA Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian cells. The system is designed around the high affinity and selectivity of Ni-NTA Agarose for recombinant fusion proteins that are tagged with six tandem histidine residues.

The Ni-NTA Purification System is a complete system that includes purification buffers and resin for purifying proteins under native, denaturing, or hybrid conditions. The resulting proteins are ready for use in many target applications.

This manual is designed to provide generic protocols that can be adapted for your particular proteins. The optimal purification parameters will vary with each protein being purified.
 
Ni-NTA Resin

Ni-NTA Agarose is used for purification of recombinant proteins expressed in bacteria, insect, and mammalian cells from any 6xHis-tagged vector. The resin exhibits high affinity and selectivity for 6xHis-tagged recombinant fusion proteins.

Proteins can be purified under native, denaturing, or hybrid conditions using the Ni-NTA Agarose. Proteins bound to the resin are eluted with low pH buffer or by competition with imidazole or histidine. The resulting proteins are ready for use in target applications.
 
The protocols provided in this manual are generic, and may not result in 100% pure protein. These protocols should be optimized based on the binding characteristics of your particular proteins.
 
Binding Characteristics

Ni-NTA Agarose uses nitrilotriacetic acid (NTA), a tetradentate chelating ligand, in a highly cross-linked 6% agarose matrix. NTA binds Ni2+ ions by four coordination sites.
 
Native Versus Denaturing Conditions

The decision to purify 6xHis-tagged proteins under native or denaturing conditions depends on the solubility of the protein and the need to retain biological activity for downstream applications.

  • Use native conditions if your protein is soluble (in the supernatant after lysis) and you want to preserve protein activity.
  • Use denaturing conditions if the protein is insoluble (in the pellet after lysis) or if your downstream application does not depend on protein activity.
  • Use hybrid protocol if your protein is insoluble but you want to preserve protein activity. Prepare the lysate and columns under denaturing conditions and then use native buffers during the wash and elution steps to refold the protein. Note that this protocol may not restore activity for all proteins.
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Preparing Cell Lysates

Introduction

Instructions for preparing lysates from bacteria, insect, and mammalian cells using native or denaturing conditions are described below.

Materials Needed

You will need the following items:

  • Native Binding Buffer for preparing lysates under native conditions
  • Sonicator
  • 10 μg/ml RNase and 5 μg/ml DNase I (optional)
  • Guanidinium Lysis Buffer (supplied with the system) for preparing lysates under denaturing conditions
  • 18-gauge needle
  • Centrifuge
  • Sterile, distilled water
  • SDS-PAGE sample buffer
  • Lysozyme for preparing bacterial cell lysates
  • Bestatin or leupeptin, for preparing mammalian cell lysates

Processing Higher Amount of Starting Material

Instructions for preparing lysates from specific amount of starting material (bacteria, insect, and mammalian cells) and purification using 2 ml resin under native or denaturing conditions are described in this manual. If you wish to purify your protein of interest from higher amounts of starting material, you may need to optimize the lysis protocol and purification conditions (amount of resin used for binding). The optimization depends on the expected yield of your protein and amount of resin to use for purification. Perform a pilot experiment to optimize the purification conditions and then based on the pilot experiment results, scale-up accordingly.


Preparing Bacterial Cell Lysate—Native Conditions

Follow the procedure below to prepare bacterial cell lysate under native conditions. Scale up or down as necessary.

  1. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for 5 minutes in a Sorvall SS-34 rotor). Resuspend the cells in 8 ml of Native Binding Buffer.

  2. Add 8 mg lysozyme and incubate on ice for 30 minutes.

  3. Using a sonicator equipped with a microtip, sonicate the solution on ice using six 10-second bursts at high intensity with a 10-second cooling period between each burst. Alternatively, sonicate the solution on ice using two or three 10-second bursts at medium intensity, then flash freeze the lysate in liquid nitrogen or a methanol dry ice slurry. Quickly thaw the lysate at 37°C and perform two more rapid sonicate-freeze-thaw cycles.

  4. Optional: If the lysate is very viscous, add RNase A (10 μg/ml) and DNase I (5 μg/ml) and incubate on ice for 10–15 minutes. Alternatively, draw the lysate through a 18-gauge syringe needle several times.

  5. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular debris. Transfer the supernatant to a fresh tube. Note: Some 6xHis-tagged protein may remain insoluble in the pellet, and can be recovered by preparing a denatured lysate followed by the denaturing purification protocol. To recover this insoluble protein while preserving its biological activity, you can prepare the denatured lysate and then follow the hybrid protocol. Note that the hybrid protocol may not restore activity in all cases, and should be tested with your particular protein.

  6. Remove 5 μl of the lysate for SDS-PAGE analysis. Store the remaining lysate on ice or freeze at -20°C. When ready to use, proceed to the protocol below.

Preparing Bacterial Cell Lysate — Denaturing Conditions

Follow the procedure below to prepare bacterial cell lysate under denaturing conditions:

  1. Equilibrate the Guanidinium Lysis Buffer, pH 7.8 (supplied with the system) to 37°C.

  2. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for 5 minutes in a Sorvall SS-34 rotor).

  3. Resuspend the cell pellet in 8 ml of Guanidinium Lysis Buffer from Step 1.

  4. Slowly rock the cells for 5–10 minutes at room temperature to ensure thorough cell lysis.

  5. Sonicate the cell lysate on ice with three 5-second pulses at high intensity.

  6. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular debris. Transfer the supernatant to a fresh tube.

  7. Remove 5 μl of the lysate for SDS-PAGE analysis. Store the remaining lysate on ice or at -20°C. When ready to use, proceed to the denaturing protocol or hybrid protocol. Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCA precipitation prior to SDS-PAGE to prevent the precipitation of SDS.

Harvesting Insect Cells

For detailed protocols dealing with insect cell expression, consult the manual for your particular system. The following lysate protocols are for baculovirusinfected cells and are intended to be highly generic. They should be optimized for your cell lines. For baculovirus-infected insect cells, when the time point of maximal expression has been determined, large scale protein expression can be carried out. Generally, the large-scale expression is performed 1 liter flasks seeded with cells at a density of 2 × 106 cells/ml in a total volume of 500 ml and infected with high titer viral stock at an MOI of 10 pfu/cell. At the point of maximal expression, harvest cells in 50 ml aliquots  Pellet the cells by centrifugation and store at -70°C until needed. Proceed to preparing cell lysates using native or denaturing conditions as described below.

Preparing Insect Cell Lysate—Native Condition

  1. Prepare 8 ml Native Binding Buffer containing leupeptin (a protease inhibitor) at a concentration of 0.5 μg/ml.

  2. After harvesting the cells, resuspend the cell pellet in 8 ml Native Binding Buffer containing 0.5 μg/ml Leupeptin.

  3. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dry ice/ethanol bath and a 42 C water bath.

  4. Shear DNA by passing the preparation through an 18-gauge needle four times.

  5. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular debris. Transfer the supernatant to a fresh tube.

  6. Remove 5 μl of the lysate for SDS-PAGE analysis. Store remaining lysate on ice or freeze at -20° C.

Preparing Insect Cell Lysate — Denaturing Condition

  1. After harvesting insect cells, resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer (supplied with the system).

  2. Pass the preparation through an 18-gauge needle four times.

  3. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular debris. Transfer the supernatant to a fresh tube.

  4. Remove 5 μl of the lysate for SDS-PAGE analysis. Store remaining lysate on ice or freeze at -20° C. When ready to use, proceed to the denaturing protocol or hybrid protocol. Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCA precipitation prior to SDS-PAGE to prevent the precipitation of SDS.

Preparing Mammalian Cell Lysate—Native Conditions

For detailed protocols dealing with mammalian expression, consult the manual for your particular system. The  following protocols are intended to be highly generic, and should be optimized for your cell lines. To produce recombinant protein, you need between 5 x 106 and 1 x 107 cells. Seed cells and grow in the appropriate mediu  until they are 80–90% confluent. Harvest the cells by trypsinization. You can freeze the cell pellet in liquid nitrogen and store at -70°C until use.

  1. Resuspend the cell pellet in 8 ml Native Binding Buffer. The addition of protease inhibitors such as bestatin and leupeptin may be necessary depending on the cell line and expressed protein.

  2. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dry ice/ethanol bath and a 42°C water bath.

  3. Shear the DNA by passing the preparation through an 18-gauge needle four times.

  4. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular debris. Transfer the supernatant to a fresh tube.

  5. Remove 5 μl of the lysate for SDS-PAGE analysis. Store the remaining lysate on ice or freeze at -20° C. Whe  ready to use, proceed to the protocol below.

Preparing Mammalian Cell Lysates — Denaturing Conditions

For detailed protocols dealing with mammalian expression, consult the manual for your particular system. The following protocols are intended to be highly generic, and should be optimized for your cell lines. To produce recombinant protein, you need between 5 x 106 and 1 x 107 cells. Seed cells and grow in the appropriate mediu  until they are 80–90% confluent. Harvest the cells by trypsinization. You can freeze the cell pellet in liquid nitrogen and store at -70°C until use.

  1. Resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer (supplied with the system).

  2. Shear the DNA by passing the preparation through an 18-gauge needle four times.

  3. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular debris. Transfer the supernatant to a fresh tube.

  4. Remove 5 μl of the lysate for SDS-PAGE analysis. Store the remaining lysate on ice or freeze at -20° C until use. When ready to use, proceed to the denaturing protocol or hybrid protocol. Note:  To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCA precipitation prior to SDS-PAGE to prevent the precipitation of SDS.
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Purification Procedure—Native Conditions

Introduction

In the following procedure, use the prepared Native Binding, Wash, and Elution Buffers, columns, and cell lysate prepared under native conditions for purification. Be sure to check the pH of your buffers before starting.

Buffers for Native Purification

All buffers for purification under native conditions are prepared from the 5X Native Purification Buffer supplied with the system. Dilute and adjust the pH of the 5X Native Purification Buffer to create 1X Native Purification Buffer. From this, you can create the following buffers:

  • Native Binding Buffer
  • Native Wash Buffer
  • Native Elution Buffer

The recipes described in this section will create sufficient buffers to perform one native purification using one kit-supplied purification column. Scale up accordingly.

Materials Needed

You will need the following items:

  • 5X Native Purification Buffer (supplied with the system).
  • 3 M Imidazole (supplied with the system)
  • NaOH
  • HCl
  • Sterile distilled water
  • Prepared Ni-NTA columns with native buffers
  • Lysate prepared under native conditions

Imidazole Concentration in Native Buffers

Imidazole is included in the Native Wash and Elution buffers to minimize the binding of untagged, contaminating proteins and increase the purity of the target protein with fewer wash steps. Note that, if your level of contaminating proteins is high, you may add imidazole to the Native Binding Buffer. If your protein does not bind well under these conditions, you can experiment with lowering or eliminating the imidazole in the buffers and increasing the number of wash and elution steps.


1X Native Purification Buffer

To prepare 100 ml 1X Native Purification Buffer, combine:

  • 80 ml of sterile distilled water
  • 20 ml of 5X Native Purification Buffer (supplied with the system)

Mix well and adjust pH to 8.0 with NaOH or HCl.

Native Binding Buffer

Without Imidazole


Use 30 ml of the 1X Native Purification Buffer (see above for recipe) for use as the Native Binding Buffer (used for column preparation, cell lysis, and binding).

With Imidazole (Optional):

You can prepare the Native Binding Buffer with imidazole to reduce the binding of contaminating proteins. (Note that some His-tagged proteins may not bind under these conditions.). To prepare 30 ml Native Binding Buffer with 10 mM imidazole, combine:

  • 30 ml of 1X Native Purification Buffer
  • 100 μl of 3 M Imidazole, pH 6.0

Mix well and adjust pH to 8.0 with NaOH or HCl.

Native Wash Buffer

To prepare 50 ml Native Wash Buffer with 20 mM imidazole, combine:

  • 50 ml of 1X Native Purification Buffer
  • 335 μl of 3 M Imidazole, pH 6.0

Mix well and adjust pH to 8.0 with NaOH or HCl.

Native Elution Buffer

To prepare 15 ml Native Elution Buffer with 250 mM imidazole, combine:

  • 13.75 ml of 1X Native Purification Buffer
  • 1.25 ml of 3 M Imidazole, pH 6.0

Mix well and adjust pH to 8.0 with NaOH or HCl.

Note: Do not use strong reducing agents such as DTT with Ni-NTA Agarose columns. DTT reduces the nickel ions in the resin. In addition, do not use strong chelating agents such as EDTA or EGTA in the loading buffers or wash buffers, as these will strip the nickel from the columns. Be sure to check the pH of your buffers before starting.

Preparing Ni-NTA Column

When preparing a column as described below, make sure that the snap-off cap at the bottom of the column remains intact. To prepare a column:

  1. Resuspend the Ni-NTA Agarose in its bottle by inverting and gently tapping the bottle repeatedly.

  2. Pipet or pour 1.5 ml of the resin into a 10-ml Purification Column. Allow the resin to settle completely by gravity (5–10 minutes) or gently pellet it by low-speed centrifugation (1 minute at 800 × g). Gently aspirate the supernatant.

  3. Add 6 ml sterile, distilled water and resuspend the resin by alternately inverting and gently tapping the column.

  4. Allow the resin to settle using gravity or centrifugation as described in Step 2, and gently aspirate the supernatant.

  5. For purification under Native Conditions, add 6 ml Native Binding Buffer.

  6. Resuspend the resin by alternately inverting and gently tapping the column.

  7. Allow the resin to settle using gravity or centrifugation as described in Step 2, and gently aspirate the supernatant.

  8. Repeat Steps 5 through 7.

Storing Prepared Columns

To store a column containing resin, add 0.02% azide or 20% ethanol as a preservative and cap or parafilm the column. Store at room temperature.

Purification Under Native Conditions

Using the native buffers, columns and cell lysate, follow the procedure below to purify proteins under native conditions:

  1. Add 8 ml lysate prepared under native conditions to a prepared Purification Column.

  2. Bind for 30–60 minutes using gentle agitation to keep the resin suspended in the lysate solution.

  3. Settle the resin by gravity or low speed centrifugation (800 × g), and carefully aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE analysis.

  4. Wash with 8 ml Native Wash Buffer. Settle the resin by gravity or low speed centrifugation (800 × g), and carefully aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE analysis.

  5. Repeat Step 4 three more times.

  6. Clamp the column in a vertical position and snap off the cap on the lower end. Elute the protein with 8–12 ml  Native Elution Buffer  Collect 1 ml fractions and analyze with SDS-PAGE.

Note:
Store the eluted fractions at 4°C. If -20°C storage is required, add glycerol to the fractions. For long term storage, add protease inhibitors to the fractions.

If you wish to reuse the resin to purify the same recombinant protein, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable binding buffer. 
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Purification Procedure—Denaturing Conditions

Introduction

Instructions to perform purification using denaturing conditions with prepared denaturing Buffers, columns, and cell lysate are described below. Be sure to check the pH of your buffers before starting.

Materials Needed


You will need the following items:

  • Denaturing Binding Buffer (supplied with the system)
  • Denaturing Wash Buffer, pH 6.0 (supplied with the system) and Denaturing Wash Buffer, pH 5.3 (see recipe below)
  • Denaturing Elution Buffer (supplied with the system)
  • Prepared Ni-NTA Agarose with denaturing buffers (below)
  • Lysate prepared under denaturing conditions

Be sure to check the pH of your buffers before starting. Note that the denaturing buffers containing urea will become more basic over time.

Preparing the Denaturing Wash Buffer pH 5.3

Using a 10 ml aliquot of the kit-supplied Denaturing Wash Buffer (pH 6.0), adjust the pH to 5.3 using HCl. Use this  for the Denaturing Wash Buffer pH 5.3 in Step 5 below.

Preparing Ni-NTA Column

When preparing a column as described below, make sure that the snap-off cap at the bottom of the column remains intact. If you are reusing the Ni-NTA Agarose, see recharging protocol.

To prepare a column:

  1. Resuspend the Ni-NTA Agarose in its bottle by inverting and gently tapping the bottle repeatedly.

  2. Pipet or pour 2 ml of the resin into a 10-ml Purification Column supplied with the kit. Allow the resin to settle completely by gravity (5-10 minutes) or gently pellet it by low-speed centrifugation (1 minute at 800 × g). Gently aspirate the supernatant.

  3. Add 6 ml of sterile, distilled water and resuspend the resin by alternately inverting and gently tapping the column.

  4. Allow the resin to settle using gravity or centrifugation as described in Step 2, and gently aspirate the supernatant.

  5. For purification under Denaturing Conditions, add 6 ml of Denaturing Binding Buffer.

  6. Resuspend the resin by alternately inverting and gently tapping the column.

  7. Allow the resin to settle using gravity or centrifugation as described in Step 2, and gently aspirate the supernatant. Repeat Steps 5 through 7.

Purification Under Denaturing Conditions

Using the denaturing buffers, columns, and cell lysate, follow the procedure below to purify proteins under denaturing conditions:

  1. Add 8 ml lysate to a prepared Purification Column.

  2. Bind for 15–30 minutes at room temperature using gentle agitation (e.g., using a rotating wheel) to keep the resin suspended in the lysate solution. Settle the resin by gravity or low speed centrifugation (800 × g), and carefully aspirate the supernatant.

  3. Wash the column with 4 ml Denaturing Binding Buffer by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 × g), and carefully aspirate the supernatant.  Save supernatant at 4º C for SDS-PAGE analysis. Repeat this step one more time.

  4. Wash the column with 4 ml Denaturing Wash Buffer (pH 6.0) by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 × g), and carefully aspirate the supernatant. Save supernatant at 4º C for SDS-PAGE analysis. Repeat this step one more time.

  5. Wash the column with 4 ml Denaturing Wash Buffer pH 5.3 (see above) by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 × g), and carefully aspirate the supernatant. Save supernatant at 4º C for SDSPAGE analysis. Repeat this step once more for a total of two washes with Denaturing Wash Buffer pH 5.3.

  6. Clamp the column in a vertical position and snap off the cap on the lower end. Elute the protein by adding 5 ml Denaturing Elution Buffer. Collect 1 ml fractions and monitor the elution by taking OD280 readings of the fractions. Pool fractions that contain the peak absorbance and dialyze against 10 mM Tris, pH 8.0, 0.1% Triton X-100 overnight at 4°C to remove the urea. Concentrate the dialyzed material by any standard method (i.e., using 10,000 MW cut-off, low-protein binding centrifugal instruments or vacuum concentration instruments). If you wish to reuse the resin to purify the same recombinant protein, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable binding buffer.
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Purification Procedure—Hybrid Conditions

Introduction

For certain insoluble proteins, the following protocol can be used to restore protein activity following cell lysis and binding under denaturing conditions. Note that this procedure will not work for all proteins, and should be tested using your particular recombinant proteins. Be sure to check the pH of your buffers before starting. Note that the denaturing buffers containing urea will become more basic over time.

Materials Needed

You will need the following items:

  • Denaturing Binding Buffer (supplied with the system)
  • Denaturing Wash Buffer, pH 6.0 (supplied with the system)
  • Native Wash Buffer
  • Native Elution Buffer
  • Prepared Ni-NTA Agarose Columns under denaturing conditions
  • Lysate prepared under denaturing conditions Ni-NTA Columns Prepare the Ni-NTA columns using Denaturing Binding Buffer.

Purification Under Hybrid Conditions

Using the denaturing buffers and columns and cell lysate prepared under denaturing conditions, follow the purification procedure below:

  1. Add 8 ml lysate to a prepared Purification Column.

  2. Bind for 15–30 minutes at room temperature using gentle agitation (e.g., on a rotating wheel) to keep the resin suspended in the lysate solution. Settle the resin by gravity or low speed centrifugation (800 × g) and carefully aspirate the supernatant.

  3. Wash the column with 4 ml Denaturing Binding Buffer by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 × g) and carefully aspirate the supernatant. Save supernatant at 4º C for SDS-PAGE analysis. Repeat this step one more time.

  4. Wash the column with 4 ml Denaturing Wash Buffer (pH 6.0) by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 × g) and carefully aspirate the supernatant. Save supernatant at 4º C for SDS-PAGE analysis. Repeat this step one more time.

  5. Wash the column with 8 ml Native Wash Buffer by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 × g) and carefully aspirate the supernatant. Save supernatant at 4º C for SDS-PAGE analysis. Repeat this step three more times for a total of four native washes.

  6. Clamp the column in a vertical position and snap off the cap on the lower end. Elute the protein with 8–12 ml Native Elution Buffer. Collect 1 ml fractions and analyze with SDS-PAGE. If you wish to reuse the resin to purify the same recombinant protein, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable binding buffer.

Troubleshooting

Introduction

Review the information below to troubleshoot your experiments with the Ni-NTA Purification System. For troubleshooting problems with antibody detection, see the antibody manual supplied with the system.

Problem Probable Cause Possible Solution
No recombinant protein recovered following elution.Nothing bound because of protein “folding.”Try denaturing conditions.
 Expression levels too low.Optimize expression levels using the guidelines in your expression manual.
 Protein washed out by too stringent washing.Raise pH of wash buffer in high-stringency wash step. Wash less extensively in high stringency wash step.
 Not enough sample loaded.Increase amount of sample loaded or lysate used.
 Recombinant protein has very high affinity for Ni-NTA Agarose.Increase stringency of elution by decreasing the pH or increasing the imidazole concentration. To preserve activity, use EDTA or EGTA (10– 100 mM ) to strip resin of nickel ions and elute protein.
 Protein degraded.Perform all purification steps at 4°C. Check to make sure that the 6xHis-tag is not cleaved during processing or purification. Include protease inhibitors during cell lysis.
Some recombinant protein is in the flow through and wash fractions.Protein overload.Load less protein on the column or use more resin for purification.
Good recombinant protein recovery but contaminated with non-recombinant proteins.Wash conditions not stringent enough.Lower pH of wash buffer in high-stringency wash step. Wash more extensively.
 Other His-rich proteins in sampleConsider an additional high stringency wash at a lower pH (i.e., between pH 6 and pH 4) before the elution step.

Further purify the eluate on a new Ni-NTA Agarose column after dialysis of the eluate against the binding buffer and equilibrating the column with binding buffer.

Perform second purification over another type of column.

Recombinant protein has low
affinity for resin; comes off in
wash with many
contaminating proteins.
Try denaturing conditions.

Try an imidazole step gradient elution.

Try a pH gradient with decreasing pH.
Low recombinant
protein recovery and
contaminated with
non-recombinant
proteins.
Recombinant protein not
binding tightly to resin.
Try denaturing conditions.
Try “reverse-chromatography”: bind lysate,
including recombinant protein; allow
recombinant protein to come off in low
stringency washes; collect these fractions; redo chromatography on saved fractions on
new or stripped and recharged column.
Works for native purification only.

Expression levels too low.
Consider an additional high stringency wash
at a lower pH (i.e., between pH 6 and pH 4)
before elution step
Column turns reddish
brown.
DTT is present in buffers.
Use β-mercaptoethanol as a reducing agent.
Column turns white.
Chelating agents present in
buffer that strip the nickel ions
from the column.
Recharge the column
Protein precipitates during binding.
Temperature is too low.
Perform purification at room temp.

Protein forms aggregates.
Add solubilization reagents such as 0.1%
Triton X-100 or Tween-20 or stabilizers such as Mg2+. These may be necessary in all buffers to maintain protein solubility.
Run column in drip mode to prevent protein from dropping out of solution.

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Recipes

Buffer Stock Solutions (10X)

To prepare the buffer solutions described below, you need to prepare sodium phosphate stock solutions:

Stock Solution A (10X)

200 mM sodium phosphate, monobasic (NaH2PO4)
5 M NaCl
Dissolve 27.6 g sodium phosphate, monobasic (NaH2PO4) and 292.9 g NaCl in 900 ml of deionized water. Mix well and adjust the volume to 1 L with deionized water. Store solution at room temperature.

Stock Solution B (10X)

200 mM sodium phosphate, dibasic (Na2HPO4)
5 M NaCl
Dissolve 28.4 g sodium phosphate, dibasic (Na2HPO4) and 292.9 g of NaCl in 900 ml of deionized water. Mix well and adjust the volume to 1 L with deionized water. Store solution at room temperature.

5X Native Purification Buffer

250 mM NaH2PO4, pH 8.0
2.5 M NaCl

Prepare 200 ml solution as follows:

  1. To 180 ml deionized water, add Sodium phosphate, monobasic 7 g NaCl 29.2 g

  2. Mix well and adjust the pH with NaOH to pH 8.0.

  3. Bring the final volume to 200 ml with water.

  4. Store buffer at room temperature.

3 M Imidazole pH 6.0

3 M Imidazole 500 mM NaCl 20 mM Sodium Phosphate Buffer, pH 6.0 Prepare 100 ml solution as follows:

  1. To 90 ml deionized water, add Imidazole 20.6 g Stock Solution A (10X) 8.77 ml,   Stock Solution B (10X) 1.23 ml

  2. Mix well and adjust the pH to 6.0 with HCl or NaOH as necessary.

  3. Bring the final volume to 100 ml with water. If the solution forms a precipitate, heat solution until the precipitate dissolves.

  4. Store buffer at room temperature.

Guanidinium Lysis Buffer

6 M Guanidine Hydrochloride
20 mM Sodium Phosphate, pH 7.8
500 mM NaCl

Prepare 100 ml solution as follows:

  1. To 90 ml deionized water, add Stock Solution A (10X) 0.58 ml,    Stock Solution B (10X) 9.42 ml,   Guanidine Hydrochloride 57.3 g

  2. Stir the solution until completely dissolved. Adjust the pH to 7.8 using 1 N NaOH or 1 N HCl.

  3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 μm filter (autoclaving the solution will alter the pH of the buffer).

  4. Store buffer at room temperature.

Denaturing Binding Buffer

8 M Urea
20 mM Sodium Phosphate pH 7.8
500 mM NaCl

Prepare 100 ml solution as follows:

  1. To 90 ml deionized water, add:  Stock Solution A (10X) 0.58 ml,  Stock Solution B (10X) 9.42 ml,  Urea 48.1g

  2. Stir the solution with gentle heating (50-60°C, do not overheat) until completely dissolved. When cooled to room temperature, adjust the pH to 7.8 using 1 N NaOH or 1 N HCl.

  3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 μm filter (autoclaving the solution will alter the pH of the buffer).

  4. Store buffer at room temperature.

Denaturing Wash Buffer

8 M Urea
20 mM Sodium Phosphate, pH 6.0
500 mM NaCl

Prepare 100 ml solution as follows:

  1. To 90 ml deionized water, add:  Stock Solution A (10X) 7.38 ml,  Stock Solution B (10X) 2.62 ml,  Urea 48.1g

  2. Stir the solution with gentle heating (50-60°C, do not overheat) until completely dissolved. Adjust the pH to 6.0 using 1 N NaOH or 1 N HCl.

  3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 μm filter (autoclaving the solution will alter the pH of the buffer).

  4. Store buffer at room temperature.

Denaturing Elution Buffer

8 M Urea
20 mM Sodium Phosphate, pH 4.0
500 mM NaCl

Prepare 100 ml as follows:

  1. To 90 ml deionized water, add:  Stock Solution A (10X) 10 ml,  Urea 48.1g

  2. Stir the solution with gentle heating (50-60°C, do not overheat) until completely dissolved. Adjust the pH to 4.0 using 1 N NaOH or 1 N HCl.

  3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 μm filter (autoclaving the solution will alter the pH of the buffer).

  4. Store buffer at room temperature.
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References

  1. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1994) Current Protocols in Molecular Biology Vol. 1. John Wiley and Sons, New York.

  2. Blochlinger, K. and Diggelmann, H. (1984) Mol. Cell Biol. 4: 2929.

  3. Frost, E. and Williams, J. (1978) Virology 91: 39.

  4. Goeddel, D.V. ed. (1991) "Expression in Mammalian Cells." Methods in Enzymology Vol. 185. Academic

  5. Press, San Diego, California.

  6. Graham, F.L. and van der Ebb, A.J. (1973) Virology 52: 456.

  7. Kawasaki Y., et al. (2000) Science 289(5482):1194–7.

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