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Products >  Protein_Expression_and_Purification >  Protein_Learning_Center >  Learning_Resources >  Denaturing_Purification_with_TALON_Resin_Protocol_At_A_Glance

PROTEIN PURIFICATION PROTOCOLS

Denaturing Purification with TALON Resin

Introduction

Denaturants, such as 6 M guanidinium, enhance protein solubility. Because overexpressed proteins in prokaryotic systems are sometimes insoluble, it may be necessary to purify proteins under denaturing conditions.

Regardless of the conditions used and the nature of the his-tagged protein being purified, most applications will benefit from the presence of 100–500 mM NaCl in the IMAC (Immobilized Metal Affinity Chromtography) buffer. In many cases, adding glycerol or ethylene glycol neutralizes nonspecific hydrophobic interactions. Small amounts of nonionic detergent may also dissociate weakly bound species.

Denaturing Buffers

When purifying proteins under denaturing conditions, we recommend preparing buffers as indicated below.

View buffer recipes >>

  • 1X Equilibration Buffer (pH 7.4)
    50 mM sodium phosphate
    6 M guanidine-HCl
    300 mM NaCl
  • Imidazole Elution Buffer (pH 7.4)
    45 mM sodium phosphate
    5.4 M guanidine-HCl
    270 mM NaCl
    150 mM imidazole
NOTES:
  • In the presence of 6 M guanidinium, the color of TALON resin will change from a pinkish-mauve to violet due to a change in metal ion hydration in response to a change in the chaotrope.
  • After removal of the guanidinium, TALON resin will return to a pinkish-mauve color.
  • The change to violet does not reflect any change in the physical or chemical-binding properties of the resin—but can be useful for indicating the buffer in which the resin is suspended, and for following the movement of guanidinium through the resin bed.

Select a protocol:

Choose between mini-scale purification, batch/gravity-flow column purification, and large-scale purification protocols.

Protocol: Mini-Scale Denaturing Purification

General Considerations

  • Applications
    Mini-scale protein purification is ideal for any of the following:
    • Checking for a his-tagged protein
    • Determining expression levels
    • Testing buffer conditions

    IMPORTANT: This protocol is not intended for obtaining highly purified his-tagged protein samples.

  • Recommended Tubes or Spin Columns
    This procedure involves adding a small amount of TALON resin to a clarified crude cell lysate in a microfuge tube. You can also use a TALON Spin Column.
  • Analysis of Results
    We recommend that you set aside a sample after each critical step of the procedure, and analyze all samples by SDS-PAGE.

Protocol

  1. Transfer 1 ml of expression culture to a 1.5-ml microcentrifuge tube.
  2. Centrifuge at 14,000 rpm for 2 min.
  3. Remove and discard supernatant.
  4. Add 0.5 ml of Equilibration Buffer (pH 8.0).
  5. Vortex until the cell pellet is completely dissolved.

    NOTE: You may need to use lysozyme (0.75 mg/ml of native buffer) to completely disrupt the cells.

  6. Centrifuge the cell lysate from Step 5 at 14,000 rpm for 5 min to pellet any insoluble debris.
  7. Prepare 50 µl of prewashed TALON resin as follows:
    1. Thoroughly resuspend the TALON resin stock.
    2. Immediately transfer 100 µl of TALON resin suspension to a clean 1.5-ml microfuge tube.
    3. Centrifuge at 14,000 rpm for 2 min to pellet the resin.
    4. Remove and discard the supernatant. The pellet should contain about 50 µl of TALON resin.
  8. Set aside 50 µl of the cell lysate supernatant from Step 6 for later analysis and transfer the remainder to the 1.5-ml microfuge tube from Step 7.d, which contains 50 µl of prewashed TALON resin.
  9. Agitate the sample at room temperature for 10 min.
  10. Centrifuge at 14,000 rpm for 1 min to pellet protein/resin complexes.
  11. Carefully remove the supernatant and set aside 50 µl for later analysis. A high protein concentration in this sample indicates a problem with protein binding.
  12. Add 1 ml of Equilibration Buffer.
  13. Vortex for a few seconds.
  14. Centrifuge at 14,000 rpm for 1 min to pellet resin.
  15. Remove the supernatant and set aside 50 µl (“first wash”) for later analysis. Discard the remainder of the supernatant.
  16. Repeat Steps 12–15. Set aside 50 µl for analysis.
  17. Elute bound his-tagged protein by adding 50 µl of Elution Buffer to the resin/protein pellet and briefly vortexing.
  18. Centrifuge briefly at 14,000 rpm.
  19. Carefully remove the supernatant containing the his-tagged protein.
  20. Repeat Steps 16–18. Alternatively, if you only intend to determine the concentration of his-tagged protein in your sample, you can achieve a more complete elution and a more accurate protein quantification by eluting with EDTA as follows:
    1. Add 50 µl of 100 mM EDTA (pH 8.0) and vortex briefly.
    2. Centrifuge briefly at 14,000 rpm.
    3. Carefully remove the supernatant containing the his-tagged protein.
      IMPORTANT:
    1. Since EDTA removes bound metal from the resin, protein samples eluted with EDTA will contain cobalt and EDTA, which may inhibit enzyme activity as well as cause the protein to precipitate.
    2. TALON resin cannot be reused with the same protein unless it is fully washed and re-equilibrated—and cannot be reused with a different protein unless it is completely regenerated.
  21. Add 12 µl of 5X SDS-PAGE Sample Buffer to each of the saved samples.

    NOTE: The sample buffer will reduce multimers to monomers; thus, only a single band will be visible on an SDS-PAGE gel, even for naturally homologous multimeric proteins.

  22. Heat samples at 95–98°C for 5 min.
  23. Load samples and analyze on an SDS-PAGE gel.

Protocol: Batch/Gravity-Flow Column Denaturing Purification

General Considerations

For IMAC columns using TALON resin, we recommend a hybrid batch/gravity-flow procedure. This method combines the speed and convenience of a batch procedure with the higher purity of the gravity-flow column method.

  • The binding and initial washing steps are performed in a batch format to save time, eliminate extraneous debris, and avoid column clogging
  • After the initial washes, the resin is transferred to a column for additional washing and protein elution.

Protocol

  1. Estimating Target Protein Expression Level
  2. If this is the first time you have prepared clarified samples from cells expressing a particular recombinant protein, we recommend that you estimate the protein’s expression level in that host strain as follows before preparing your sample:

    1. Perform a mini-scale purification (see the Mini-Scale Native Purification Protocol, above), and then analyze a portion by SDS-PAGE in parallel with protein standards.
    2. Once satisfactory expression is observed, proceed with sample preparation.
  3. Sample Preparation to Isolate Denatured Proteins
  4. This procedure can be used with any TALON resin or TALON Superflow resin. It has been optimized for extraction of denatured proteins from fresh or frozen cell pellets.

    1. Harvest 20–25 ml of cell culture by centrifugation at 1,000–3,000 x g for 15 min at 4°C.
    2. Resuspend pellet in 2 ml of denaturing Equilibration Buffer per 20–25 ml of culture.
    3. Gently agitate or stir the sample until it becomes translucent.
    4. Centrifuge the sample at 10,000–12,000 x g for 20 min at 4°C to pellet any insoluble material.
    5. Carefully transfer the supernatant (the clarified sample) to a clean tube without disturbing the pellet.
    6. Set aside a small portion of the clarified sample for SDS/PAGE analysis before starting purification.

      NOTE: Samples containing 6 M guanidinium must be dialyzed overnight against buffer containing 8 M urea before loading on a gel.

    7. The crude lysate from Step e can be applied directly to a TALON CellThru column without centrifugation. If the lysate is centrifuged (10,000–12,000 x g for 20 min at 4°C), the resulting supernatant can be applied to any column containing TALON or TALON Superflow resin.
  5. Resin Equilibration
    1. Thoroughly resuspend the TALON, TALON Superflow, or TALON CellThru resin.
    2. Immediately transfer the required amount of resin suspension to a sterile tube that will accommodate 10–20 times the resin bed volume.
    3. Centrifuge at 700 x g for 2 min to pellet the resin.
    4. Remove and discard the supernatant.
    5. Add 10 bed volumes of Equilibration Buffer and mix briefly to pre-equilibrate the resin.
    6. Recentrifuge at 700 x g for 2 min to pellet the resin. Discard the supernatant.
    7. Repeat Steps e and f.
  6. Sample Application
    1. Add crude lysate from Step 2.d to a TALON CellThru column or clarified sample from Step 2.e to any column containing TALON or TALON Superflow resin.
    2. Gently agitate at room temperature or on ice for 20 min on a platform shaker to allow the his-tagged protein to bind the resin.
    3. NOTE: Incubation on ice will decrease proteolysis.

    4. Centrifuge at 700 x g for 5 min. Carefully remove as much supernatant as possible without disturbing the resin pellet.
  1. Washing
    1. Wash the resin by adding 10–20 bed volumes of Equilibration Buffer. Gently agitate the suspension at room temperature or on ice for 10 min on a platform shaker for thorough washing.
    2. Centrifuge at 700 x g for 5 min.
    3. Remove and discard the supernatant.
    4. Repeat Steps a–c with 10–20 bed volumes of Equilibration Buffer.
    5. Add one bed volume of Equilibration Buffer to the resin, and resuspend by vortexing.

      NOTE: Steps f–h can be performed on ice or at room temperature, but incubation on ice will decrease proteolysis.

    6. Transfer the resin to a 2-ml gravity-flow column with an end-cap in place, and allow the resin to settle out of suspension.
    7. Remove the end-cap and allow the buffer to drain until it reaches the top of the resin bed, making sure no air bubbles are trapped in the resin bed.
    8. Wash column once with 5 bed volumes of Wash Buffer.
  2. Elution & Analysis
    1. Elute the his-tagged protein by adding 5 bed volumes of Elution Buffer to the column. Collect the eluate in 500 µl fractions.

      NOTE: Under most conditions, the majority of the his-tagged protein will be recovered in the first two bed volumes.

    2. Use spectrophotometric and SDS-PAGE analysis to determine which fraction(s) contain(s) the majority of the his-tagged protein.

      NOTE: Use a Bradford protein assay (Bradford, 1976) or UV absorbance at 280 nm.

Protocol: Large-Scale Batch Denaturing Purification

General Considerations

This method purifies his-tagged proteins faster than gravity-flow columns; however, batch washes remove impurities less efficiently than gravity-flow columns. Therefore, they require larger wash buffer volumes to obtain pure his-tagged proteins.

Protocol

  1. Estimating Target Protein Expression Level
  2. If this is the first time you have prepared clarified samples from cells expressing a particular recombinant protein, we recommend that you estimate the protein’s expression level in that host strain as follows before preparing your sample:

    1. Perform a mini-scale purification (see the Mini-Scale Native Purification Protocol, above), and then analyze a portion by SDS-PAGE in parallel with protein standards.
    2. Once satisfactory expression is observed, proceed with sample preparation.
  3. Sample Preparation to Isolate Denatured Proteins
  4. This procedure can be used with any TALON resin or TALON Superflow resin. It has been optimized for extraction of denatured proteins from fresh or frozen cell pellets.

    1. Harvest cell culture by centrifugation at 1,000–3,000 x g for 15 min at 4°C.
    2. Resuspend pellet in 2 ml of denaturing Equilibration Buffer per 20–25 ml of culture.
    3. Gently agitate or stir the sample until it becomes translucent.
    4. Centrifuge the sample at 10,000–12,000 x g for 20 min at 4°C to pellet any insoluble material.
    5. Carefully transfer the supernatant (the clarified sample) to a clean tube without disturbing the pellet.
    6. Set aside a small portion of the clarified sample for SDS/PAGE analysis before starting purification.

      NOTE: Samples containing 6 M guanidinium must be dialyzed overnight against buffer containing 8 M urea before loading on a gel.

    7. The crude lysate from Step e can be applied directly to a TALON CellThru column without centrifugation. If the lysate is centrifuged (10,000–12,000 x g for 20 min at 4°C), the resulting supernatant can be applied to any column containing TALON or TALON Superflow resin.
  5. Resin Equilibration
    1. Thoroughly resuspend the TALON, TALON Superflow, or TALON CellThru resin.
    2. Transfer the required amount of resin to a glass filter with a pore size of 10–20 µm.
    3. Apply a vacuum to the filter to remove excess ethanol.
    4. Add 5 bed volumes of deionized water to the resin and apply vacuum.
    5. Add 5 bed volumes of Equilibration Buffer to the resin and apply vacuum.
    6. Repeat Step e two times.
  6. Sample Application
    1. Add crude lysate from Step 2.d to a TALON CellThru column or clarified sample from Step 2.e to any column containing TALON or TALON Superflow resin.
    2. Apply vacuum and collect the filtrate.
  7. Washing
    1. Incubate the suspension at room temperature for 10 min with periodic stirring to promote thorough washing.
    2. Apply vacuum to remove buffer.
    3. Repeat the above wash (Steps a–b) 2–3 times with 10–20 bed volumes of Equilibration Buffer.
    4. [Optional]: If necessary, repeat Step c under more stringent conditions using 5 mM imidazole in Equilibration Buffer.
  8. Elution & Analysis
    1. Elute the his-tagged protein by adding 5 bed volumes of Elution Buffer.
    2. Gently agitate the suspension at room temperature for 5 min.
    3. Apply vacuum, and collect the purified his-tagged protein.
    4. Repeat Steps a–c two times, collecting separate fractions.
    5. Use spectrophotometric analysis (absorbance at 280 nm) or a Bradford protein assay and SDS-PAGE analysis to determine which fraction(s) contain(s) the majority of the his-tagged protein.

Download the Mini-Scale Purification Protocol PDF >>

Download the Batch/Gravity-Flow Column Purification Protocol PDF >>

Download the Large-Scale Purification Protocol PDF >>

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