GE Healthcare

His MicroSpin Purification Module

Product Booklet

Code: 27-4770-01

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Page finder1. Legal 3

2. Handling 4 2.1. Safety warnings and precautions 4 2.2. Storage 4 2.3. Quality control 4

3. Components of the kit 5

4. Materials not supplied 6

5. Introduction 7

6. Protocols 9 6.1. Overview 9 6.2. Essential preliminaries 10 6.3. Culture growth and lysis 11 6.4. Use with MicroPlex 24 Vacuum 13 6.5. Use with a microcentrifuge 16 6.6. Use with MicroPlex 24 (Spin) 18 6.7. Analysis of samples collected during purification 20

7. Appendixes 21 7.1. Characteristics of His MicroSpin columns 21 7.2. Optimization of IMAC purification 21

8. Troubleshooting 24

9. References 29

10. Companion products available from GE Healthcare 30

1. LegalGE and GE monogram are trademarks of General Electric Company.

HisTrap, HiTrap, MicroPlex, MicroSpin and Sepharose are trademarks of GE Healthcare companies.

Kathon is a trademark of Rohm and Haas Company.

Triton is a trademark of Union Carbide Chemicals and Plastics Co.

Coomassie is a trademark of ICI Americas, Inc.

© 2006 General Electric Company – All rights reserved.

GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specification and features shown herein, or discontinue the product described at any time without notice or obligation.

Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions.

http//www.gehealthcare.com/lifesciences

GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK

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2. Handling

2.1. Safety warnings and precautionsWarning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.

All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice.

2.2. StorageStore all components at 4°C. Do not freeze.

2.3. Quality controlThe Chelating Sepharose™ Fast Flow resin used in the His MicroSpin™ Purification Module is tested for its ability to purify ahis-tagged protein.

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3. Components of the kitSufficient reagents are supplied to purify up to 5 mg of his-tagged fusion protein.

The following components require preparation before use. Refer to “Essential preliminaries” (page 10) for more details.

10 x PBS 1.4 M NaCl, 27 mM KCl, 101 mM Na2HPO4, 18 mM KH2PO4 (pH 7.3).

Imidazole 25 ml of 4 M imidazole.elution buffer CAUTION: Avoid contact with skin or eyes.

Phosphate/NaCl 8 x stock solution: 160 mM buffer (PN) phosphate, 4 M NaCl, pH 7.4.

IPTG Isopropyl β-D-thiogalactoside, 500 mg.

His MicroSpin 50 MicroSpin columns eachcolumns* containing 0.1 ml of a 50% slurry of Ni-charged Chelating Sepharose™ Fast Flow in 5x Phosphate/NaCl + 20 mM imidazole + 0.05% Kathon™ CG/ICP Biocide.

* Store His MicroSpin columns at 4°C. Do not freeze.

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4. Materials not supplied• Bottle—500–1000 ml for dilution of PBS.

• Reagents, equipment and gels—for SDS-PAGE

• 2x YTA medium— Tryptone 16 g/l Yeast extract 10 g/l NaCl 5 g/l

Dissolve above ingredients in 900 ml of distilled H2O. Adjust the pH to 7.0 with NaOH. Adjust the volume to 1 litre. Sterilize by autoclaving for 20 minutes. Once the medium has cooled, aseptically add 1 ml of a 100 mg/ml ampicillin stock solution (final concentration 100 μg/ml). To prepare as a solid medium, add 12–15 g of agar prior to autoclaving.

• 6x SDS loading buffer (10)—0.35 M Tris-HCl (pH 6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol

(or 5% β-mercaptoethanol), 0.012% (w/v) bromophenol blue. Store in 0.5 ml aliquots at -20°C.

5. IntroductionThe use of fusion tags in the purification and detection of recombinant proteins has become a common practice (1). In addition to large protein tags such as glutathione S-transferase, some fusion vectors encode smaller peptide tags. Tags that include several histidine (his) residues in close proximity to each other can be purified by virtue of the strong interaction of the histidines with divalent metal ions (2–5). When the metal ions are strongly bound to a support matrix, such as Chelating Sepharose™ Fast Flow, affinity purifi cation of the his-tagged fusion proteins can be performed by immobilized metal ion adsorption chromatography (IMAC; 6, 7).

The most popular purification handle utilized in IMAC is the poly-histidine tag composed of six consecutive histidine residues [(his)6]. Expression vectors that encode polyhistidine tags are available from many commercial sources. While vectors have been developed for expression of his-tagged pro teins in a variety of systems includingbacteria, yeast, baculovirus, vaccinia, and cultured eukaryotic cells, the protocols described in this booklet address the small-scale purification of such proteins from bacterial cell lysates. The pro cedures may, however, be adapted to other related his-tag expression systems.

The His MicroSpin Purification Module contains MicroSpin columns pre-packed with Ni2+-charged Chelating Sepharose Fast Flow. These columns will efficiently capture his-tagged proteins from samples such as bacterial lysates of recombinant (his)6-vector transformants. Non-tagged proteins and any other contaminants are washed easily from the column. Weakly binding proteins that contain surface residues that interact with Ni2+ ions are washed away using low concentrations of imidazole. Strongly bound his-tagged proteins are ultimately desorbed from the columns by competitive elution with high concentrations of imidazole.

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The imidazole concentration of the wash and elution buffers needs to be optimized for each his-tagged protein since each fusion protein has unique properties that influence its purification. Furthermore, different extracts can be expected to contain different amounts of background proteins capable of binding immobilized Ni2+. The imidazole concentration that allows the greatest amount of weakly binding contaminating proteins to be washed from the column while maintaining the specific retention of the his-tagged protein of interest can be readily determined using the His MicroSpin Purification Module.

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6. Protocols

6.1. OverviewThe His MicroSpin Purification Module has been designed for screening large numbers of bacterial lysates for expression of his-tagged recombinant fusion proteins. This permits the analysis of many clones simultaneously so that those with the highest expression levels can be selected and used for large-scale preparations. This module is also useful for processing numerous samples during the optimization of expression conditions. For example, using the MicroSpin format on small samples, you may rapidly evaluate media, growth temperatures, culture densities, induction conditions and other variables to choose the best conditions for expression.

Yield of fusion protein is highly variable and is affected by the natureof the fusion protein, the host cell and the culture conditions used. Each MicroSpin column in the His MicroSpin Purification Module contains a 50 μl bed volume of Chelating Sepharose Fast Flow, enough to purify up to 100 μg of a test (his)6-tagged recombinant protein. The capacity of the matrix for other his-tagged fusion proteins may be less than that for the test protein and may be expected to vary with the nature of the fusion protein and the exact binding conditions used.

A protocol for the growth and lysis of small cultures of bacterial strains expressing his-tagged proteins is provided in Protocol 1. Protocol 2 provides instrucitons to screen large numbers of lysates using MicroPlex 24 Vacuum. The use of a microcentrifuge to process a small number of lysates is given in Protocol 3. A protocol for screening large numbers of lysates using MicroPlex 24 (Spin) is

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provided in Protocol 4. Protocol 5 describes several approaches for the analysis of affinity-purified fusion proteins.

6.2. Essential preliminariesPrior to first use, the following reagents provided in the kit should be prepared as indicated:

1 x PBS: Dilute 10 x PBS in the kit with sterile water to 1 x PBS final concentration. Store at 4°C.

Note: If a precipitate is present in the 10 x PBS, let the solution stand at room temperature, then swirl to dissolve the precipitate before diluting.

1 x PN (without imidazole): Dilute the 8 x Phosphate/NaCl buffer stock 1:8 with distilled water. This optional buffer can be used to dilute bacterial cultures.

PNI20 wash buffer: Combine 6.25 ml of 8 x Phosphate/NaCl buffer stock solution with 0.25 ml of Imidazole elution buffer. Add distilled water to a final volume of 50 ml. This solution will contain 20 mM phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4.

Note: For formulation of wash buffers containing different imidazole concentrations for optimization of IMAC purification, see Appendix 2, page 22.

PNI400 elution buffer: Combine 6.25 ml of 8 x Phosphate/NaCl buffer stock solution with 5 ml of Imidazole elution buffer. Add distilled water to a final volume of 50 ml. This solution will contain 20 mM phosphate, 0.5 M NaCl, 400 mM imidazole, pH 7.4.

IPTG solution: To prepare a 100 mM solution, dissolve the total contents of the vial containing the lyophilized IPTG in 20 ml of sterile water. Dispense as 1 ml aliquots and store at -20°C.

Additional reagents and materials may be found in “Materials not supplied,” page 6.

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Small aliquots of samples should be retained at key steps in the procedure for analysis of the purification method (Protocol 5).

6.3. Culture growth and lysisSections of this procedure have been adapted with permission from Current Protocols in Molecular Biology, Vol. 2, Supplement 10, Unit 16.7. Copyright © 1990 by Current Protocols.

Some vectors that encode his-tagged fusion proteins also carry the lacIq gene, so there are no specific host requirements for propagation of the plasmids or for expression of fusion proteins. For such plasmids, the protease-deficient host strain, E. coli BL21 [F-, ompT, hsdS (rB-, mB-), gal] (8, 9), is recommended for expression of his-tagged proteins. Vectors that encode his-tagged fusion proteins under the control of the lac promoter but do not carry the lacIq gene on the plasmid will require a lacIq host strain.

Because the available volume of a prepacked His MicroSpin column is relatively small, no more than 400 μl of culture lysate can be applied at a time. This represents the volume of lysate produced from an 8 ml culture. For lysing cultures ≤ 8 ml, the following freeze/thaw method is recommended and can be performed in standard 1.5 ml microcentrifuge tubes. Wash volumes for His MicroSpin columns may not exceed 600 μl.

Prepare kit reagents as described on page 10 before starting this procedure.

Culture growthRecipes are provided in “Materials not supplied,” page 6

2 x YTA Medium100 mM IPTG (see “Essential preliminaries,” page 10)1 x PBS (see “Essential preliminaries,” page 10)

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1. Pick several colonies of E. coli transformed with a vector that encodes a his-tagged fusion protein and transfer each colony into a separate tube or flask containing up to 8 ml of 2 x YTA medium.

Note: For comparison, it is advisable to inoculate a control tube with bacteria transformed with the parental plasmid.

2. Grow cultures with vigorous agitation at 20–37°C to an A600 of 0.6–0.8 (3–5 hours).

3. Induce his-tagged protein expression by adding 1–10 μl of100 mM IPTG for each ml of culture volume

(final concentration = 0.1–1.0 mM).

4. Continue incubation for an additional 1–2 hours.

5. Transfer the liquid cultures to labelled centrifuge tubes. If screening ≤ 1.5 ml, each induced culture may be transferred to a microcentrifuge tube.

6. Centrifuge to sediment cells (e.g., 5 seconds at full speed in a microcentrifuge) and discard supernatants. Drain pellets thoroughly and place tubes on ice.

7. Resuspend each pellet in 50 μl of ice-cold 1x PBS for each ml of culture that was centrifuged. Transfer 10 μl of these resuspended cells to labelled tubes and save for later use in SDS-PAGEanalysis (Protocol 6.5., page 20).

Note: Except where noted, keep all samples and tubes on ice.

Cell lysisFor larger-scale cultures, sonication is recommended for the lysis of cells. However, the process is only efficient for cell suspensions of greater than 2 ml, representing culture volumes of at least 40 ml. While screening of multiple samples can be performed on aliquots of such larger sonicates, the growth and processing of a high number of cultures of this size may be unwieldy. For lysing smaller cultures

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(≤ 8 ml), the freeze/thaw method below is recommended and can be performed in standard 1.5 ml microcentrifuge tubes.

Note: We recommend testing the freeze/thaw method on a single sample before processing multiple samples, to determine if the lysate will be too viscous for handling. If the lysate of the test sample obtained after step 1.11 of this procedure is too viscous for handling, then add DNase I to a final concentration of 10 μg/ml in step 8 when processing the remainder of your samples.

8. Prepare a 10 mg/ml lysozyme solution in water. Add 1 μl of lysozyme solution for each 100 μl of cell suspension (e.g., 6 μl to each tube containing 600 μl of cell suspension). Vortex tubes gently to disperse lysozyme. Allow tubes to incubate at room temperature for 5 minutes.

9. In a fume hood, prepare a dry ice bath in an ice bucket by adding dry ice and isopropanol until a slushy consistency is achieved. Prepare a warm water bath in a separate container.

10. Place tubes containing lysozyme-treated cell suspensions in the dry ice bath until cells are frozen solid (~ 20 seconds). Transfer tubes to a flotation carrier and place in the warm water bath until the suspension becomes fully liquid (~ 1 minute). Repeat freeze/thaw cycle 10 times.

11. Spin at full speed in a microcentrifuge for 10 minutes to remove insoluble material. Save a 10 μl aliquot of the insoluble material for analysis by SDS-PAGE. Transfer the supernatants to fresh tubes.

Note: If the lysate is too viscous for handling, DNase I may be added to a final concentration of 10 μg/ml in step 8 above.

6.4. Use with MicroPlex 24 VacuumDo not apply more than 400 μl of culture lysate at a time to a His MicroSpin column. The following procedure is designed to

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accommodate lysates prepared from 2–8 ml of culture.

A vacuum source capable of providing -200 mm Hg (-200 Torr), for example, a house vacuum, is required for processing large numbers of samples with the MicroPlex 24 Vacuum (27-3567-01).

Reagents requiredHis MicroSpin columns1 x PBS (see “Essential preliminaries,” page 10)PNI20 wash buffer (see “Essential preliminaries,” page 10)PNI400 elution buffer (see “Essential preliminaries,” page 10)

1. Assemble the MicroPlex 24 Vacuum according to the instructions provided with the unit.

2. Remove the caps from each His MicroSpin column. Place the columns in the MicroPlex 24 Vacuum manifold. Fill any unused holes with the plugs provided with MicroPlex 24 Vacuum.

3. Apply 400 μl of culture lysate supernatant (from Protocol 1) directly to each His MicroSpin column.

Note: If < 400 μl of clarified lysate is to be applied, adjust the volume to 400 μl with 1 x PBS or 1 x PN prior to addition to the columns.

4. Recap each column and mix by gentle repeated inversion. Allow binding to proceed at room temperature for 5–10 minutes.

5. Remove columns from the manifold. Snap off the bottom closure and remove the top cap from each column. Return columns to the MicroPlex 24 Vacuum manifold.

6. Make sure that the vacuum line stopcock is in the closed position (i.e. perpendicular to the vacuum tubing) and that the manifold is placed squarely on the gasket and collection tray.

7. Turn the vacuum supply (e.g., house vacuum) on at the source. Turn the stopcock in the vacuum tubing assembly to the open position (i.e. parallel to the tubing). After the lysates have been

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drawn through all of the columns into the collection tray, turn the stopcock to the closed position, leaving the vacuum supply on at the source.

8. Allow the vacuum pressure to dissipate for 10–15 seconds. Once the pressure has fully dissipated, remove the manifold from the collection tray and place it on a paper towel. (The manifold should separate easily from the collection tray.)

Note: If subsequent flow-through and washes are to be analyzed, each may be collected in a clean collection plate. Transfer the gasket to a clean collection plate and reassemble the MicroPlex 24 Vacuum system. If these fractions won’t be analyzed, then the collection plate may be emptied and reused for each step prior to elution.

9. Add 600 μl of PNI20 wash buffer to each His MicroSpin column. Turn the stopcock in the vacuum tubing assembly to the open position. After the buffer has been drawn through all of the columns into the collection tray, turn the stopcock to the closed position.

10. Allow the vacuum pressure to dissipate for 10–15 seconds. Once the pressure has fully dissipated, separate the manifold from the collection tray as described above, and reassemble the apparatus with a clean collection plate.

Note: Alternatively, the MicroSpin columns can be transferred to 1.5 ml micro centrifuge tubes for elution of samples using a microcentrifuge as described in Protocol 3. This eliminates the need to transfer samples from the collection tray to tubes for long-term storage and permits smaller elution volumes to be used.

Note: Additional washes with increased concentrations of imidazole may be performed to obtain a more pure product. Note, however, that increased purity may come at the cost of decreased yield. The optimal imidazole concentration will depend on the exact protein

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being purified and the composition of the extract from which it is being isolated. A pilot experiment should be performed to determine the exact washing conditions that will result in the optimal purification of your his-tagged fusion protein. See Appendix 2 (page 22) for experimental guidelines.

11. Add 200 μl of PNI400 elution buffer to each MicroSpin column and incubate at room temperature for 5–10 minutes. Turn the stopcock in the vacuum tubing assembly to the open position. After the elution buffer has been drawn through all of the columns into the collection tray, turn the stopcock to the closed position.

Note: Yields of his-tagged protein may be increased by repeating the elution step two or three times and pooling the eluates.

12. Allow the vacuum pressure to dissipate for 10–15 seconds. Once the pressure has fully dissipated, separate the manifold from the collection tray as described previously. If desired, the tray containing the eluted samples can be covered with sealing tape (e.g., Corning Costar Catalog Number 3095).

6.5. Use with a microcentrifugeDo not apply more than 400 μl of culture lysate at a time to a His MicroSpin column. The following procedure is designed to accommodate lysates prepared from 2–8 ml of culture.

Reagents requiredHis MicroSpin columns1 x PBS (see “Essential preliminaries,” page 10)PNI20 wash buffer (see “Essential preliminaries,” page 10)PNI400 elution buffer (see “Essential preliminaries,” page 10)

1. Remove (and save) the cap of each His MicroSpin column.

2. Apply 400 μl of culture lysate supernatant (from Protocol 1) directly to each His MicroSpin column.

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Note: If < 400 μl of clarified lysate is to be applied, adjust the volume to 400 μl with 1x PBS or 1x PN prior to addition to the columns.

3. Recap each column securely and mix by gentle, repeated inversion. Allow binding to proceed at room temperature for 5–10 minutes.

4. Remove (and save) the top cap and bottom closure from each column and place each column into a clean 1.5 or 2 mlmicrocentrifuge tube. Spin all of the column/tubes in a micro-centrifuge for 1 minute at 735 x g (e.g., 3000 rpm in an Eppendorf model 5415C variable speed centrifuge with an 18-positionfixed-angle rotor).

5. Discard the buffer from each microcentrifuge tube and place each His MicroSpin column into a clean, prelabelled 1.5 or 2 ml microcentrifuge tube. Apply 600 μl of PNI20 wash buffer to each column. Spin as above to wash the matrix, saving each flow-through for future analysis, if desired.

Note: Additional washes with increased concentrations of imidazole may be performed to obtain a more pure product. Note, however, that increased purity may come at the cost of decreased yield. The optimal imidazole concentration will depend on the exact protein being purified and the composition of the extract from which it is being isolated. A pilot experiment should be performed to deter-mine the exact washing conditions that will result in the optimal purification of your his-tagged fusion protein. See Appendix 2 (page 22) for experimental guidelines.

6. Replace the bottom closure securely on each His MicroSpin column. Add 100–200 μl of PNI400 elution buffer to each column. Incubate at room temperature for 5–10 minutes.

7. Remove and discard the top cap and bottom closure from each His MicroSpin column and place each column into a clean, pre-labelled 1.5 or 2 ml microcentrifuge tube. Spin all of the column/

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tubes in a microcentrifuge for 1 minute at 735 x g to collect each eluate. Save for analysis.

Note: Yields of fusion protein may be increased by repeating the elution step two or three times and pooling all eluates.

6.6. Use with MicroPlex 24 (Spin)Do not apply more than 400 μl of culture lysate at a time to a His MicroSpin column. The following procedure is designed to accommodate lysates prepared from 2–8 ml of culture.

Reagents requiredHis MicroSpin columns1 x PBS (see “Essential preliminaries,” page 10)PNI20 wash buffer (see “Essential preliminaries,” page 10)PNI400 elution buffer (see “Essential preliminaries,” page 10)

1. Assemble the MicroPlex 24 unit according to the instructionsprovided with the unit. If two MicroPlex 24 units are used, up to 48 samples can be processed simultaneously.

2. Remove the cap from each His MicroSpin column and place the columns in the MicroPlex 24 manifold(s).

3. Apply 400 μl of culture lysate supernatant (from Protocol 1) directly to each His MicroSpin column.

Note: If < 400 μl of clarified lysate is to be applied, adjust the volume to 400 μl with 1x PBS or 1 x PN prior to addition to the columns.

4. Recap each column and mix by gentle, repeated inversion. Allow binding to proceed at room temperature for 5–10 minutes.

5. Remove columns from the manifold. Snap off the bottom closure, remove the top cap from each column. Return columns to the

MicroPlex 24 manifold.

6. Centrifuge the MicroPlex 24 unit(s) for 2 minutes according to the instructions supplied with the unit.

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Note: If subsequent flow-through and washes are to be analyzed, each may be collected in a clean collection plate. If these fractions won’t be analyzed, then the collection plate may be emptied and reused for each step prior to elution.

7. Add 600 μl of PNI20 wash buffer to each His MicroSpin column.

8. Centrifuge the MicroPlex 24 unit(s) for 2 minutes according to the instructions supplied with the unit. Remove the manifold from each collection tray and place it on a clean paper towel. Reassemble each MicroPlex 24 unit with a fresh collection tray.

Note: Alternatively, the MicroSpin columns can be transferred to 1.5 ml micro centrifuge tubes for elution of samples using a microcentrifuge as described in Protocol 3. This eliminates the need to transfer samples from the collection tray to tubes for long-term storage and permits smaller elution volumes to be used.

Note: Additional washes with increased concentrations of imidazole may be performed to obtain a more pure product. Note, however, that increased purity may come at the cost of decreased yield. The optimal imidazole concentration will depend on the exact protein being purified and the composition of the extract from which it is being isolated. A pilot experiment should be performed to deter-mine the exact washing conditions that will result in the optimal purification of your his-tagged fusion protein. See Appendix 2 (page 22) for experimental guidelines.

9. Apply 100–200 μl of PNI400 elution buffer to each His MicroSpin column and incubate at room temperature for 5–10 minutes.

10. Centrifuge the MicroPlex 24 unit(s) for 2 minutes according to the instructions supplied with the unit. If desired, the tray containing the eluted samples can be covered with sealing tape (e.g., Corning Costar Catalog Number 3095).

Note: Yields of fusion protein may be increased by repeating the elution step two or three times and pooling the eluates.

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6.7. Analysis of samples collected during purificationSDS-PAGE analysis1. Transfer 10 μl aliquots of each sample to be analyzed (e.g.,

samples retained following cell resuspension, lysis, column flow- throughs, washes, eluates, etc.) to fresh tubes.

2. To each sample, add 2 μl of 6x SDS loading buffer (see “Materials not supplied,” page 6). Vortex briefly and heat for 5 minutes at 90–100°C.

3. Load the samples onto a 10–12.5% SDS-polyacrylamide gel.

4. Run the gel for the appropriate time and stain with Coomassie blue to visualize the his-tagged protein.

If the above analysis indicates that the (his)6-tagged protein has adsorbed to the Chelating Sepharose Fast Flow media, you may proceed to large-scale purification. If, on the other hand, the his-tagged protein is absent from the purified material, it may be insoluble or expressed at very low levels; refer to the trouble-shooting guide (page 24) for a discussion of these problems.

Additional analysesIf recombinants expressing his-tagged proteins cannot be identified using the methods described above, clones can also be identified by Western blot analysis using Anti-His Antibody (27-4710-01). Another alternative is to perform a functional assay, if available, specific for the protein of interest.

The relative yield of his-tagged protein can be estimated by measuring the absorbance at 280 nm. The yield of protein may also be determined by standard chromogenic methods (e.g. Lowry, BCA, Bradford, etc.).

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7.1. Appendixes

7.1. Characteristics of His MicroSpin columnsColumn matrix: Columns are prepacked with 0.1 ml of a 50% slurry of Ni-charged Chelating Sepharose Fast Flow in 5x Phosphate/NaCl + 20 mM imidazole (+ 0.05% Kathon CG/ICP Biocide).

Chelating group: Imino-diacetic acid

Binding capacity: > 100 μg recombinant (his)6-tagged test proteinper column

Average bead size: 34 μm diameter

Bead structure: Highly cross-linked spherical agarose

Spacer arm: 7 atoms

Maximum volumetric flow rate: 4 ml/minute

Recommended volumetric flow rate: 1–4 ml/minute

Maximum operating pressure: 3 bar = 43 psi = 0.3 MPa

Chemical stability: Stable in all commonly used aqueous buffers and denaturants such as 6 M guanidine hydrochloride, 8 M urea and chaotropic salts

pH stability: 2–14 (short term, for cleaning), 3–13 (long term)

Avoid exposure to: Chelating agents (e.g., EDTA, EGTA), Reducing agents (e.g., DTT, DTE)

7.2. Optimization of IMAC purificationThe purity of his-tagged proteins purified using the His MicroSpin Purification Module can sometimes be improved by adjusting the washing and elution parameters. Varying the concentration of imidazole in the wash buffer can result in higher stringency washes that remove impurities from the Ni-charged

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Chelating Sepharose Fast Flow matrix while maintaining the binding of the desired his-tagged protein.

The concentration of imidazole in the wash buffer can be optimized by preparing a series of wash buffers of increasing imidazole concentrations (see Table 1). The format of the His MicroSpin Purification Module permits the identical application of culture lysate to several MicroSpin columns followed by the washing of each column with a different wash buffer in the series. Analysis of wash buffer flow-throughs and subsequent elution buffer fractions will help determine the optimal imidazole concentration for the wash buffer that removes the most impurities from the matrix while retaining the greatest amount of desired his-tagged protein.

It should be noted that while increasing the imidazole concentration of the wash buffer may lead to increased purity, it may also decrease the yield of the desired protein. Some portion of the desired his-tagged protein may be washed from the matrix by the higher concentrations of imidazole. Depending on the planned use of the purified protein, this may not be desirable. In the pilot experiment described above, you should determine the wash conditions that result in the optimal combination of purity and yield for your specific application.

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Table 1. Mixing table for wash/elution buffer optimization.

Imidazole Phosphate/NaCl 4M Imidazole Distilledconcentration buffer (8x) elution buffer water(mM) (ml) (ml) (ml)

0 3.75 0.000 26.25020 3.75 0.150 26.10040 3.75 0.300 25.95060 3.75 0.450 25.80080 3.75 0.600 25.650100 3.75 0.750 25.500150 3.75 1.125 25.125200 3.75 1.500 24.750250 3.75 1.875 24.375300 3.75 2.250 24.000400 3.75 3.000 23.250500 3.75 3.750 22.500

To obtain a 1 x Phosphate/NaCl solution containing the indicated concen tration of imidazole, mix the volume of 8x Phosphate/NaCl buffer indicated in the table above with the corresponding volume of 4 M Imidazole elution buffer and bring the final volume to 30 ml with distilled water. Label the buffers PNIX where X is the concentration of imidazole in mM.

The resulting wash buffer(s) can be used in Protocols 2–4 either in place of or in addition to PNI20 wash buffer.

Alternative elution buffers with the above formulations can also betested and used in Protocols 2–4 in place of PNI400 elution buffer.

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Possible causes

1. Optimize expression conditions.

2. Check DNA sequences.

3. Analyze a small aliquot of an overnight culture by SDS-PAGE.

Solutions

1. Optimization of expression conditions can dramatically improve yields. Investigate the effects of host cell strain, medium compo sition, incubation temperature and induction conditions on his-tagged protein yield. Exact conditions will vary for each protein.

2. It is essential that protein-coding DNA sequences be cloned in the proper translation frame. Cloning junctions should be sequenced to verify that inserts are in-frame with the his-tag coding region.

3. Generally, a highly expressed protein will be visible by Coo massie staining when 5–10 μl of an induced culture [(A600 ≅ 1.0)] is loaded on the gel. Nontransformed E. coli cells and cells transformed with the parental vector should be run in parallel as negative and positive controls, respectively. The presence of the his-tagged protein in total cell preparations and its absence from a clarified lysate may indicate the presence of inclusion bodies (see overleaf).

8. TroubleshootingProblem: No his-tagged protein is detected by a Coomassie-Stained SDS gel of bacterial lysate.

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Possible causes

4. Check for expression by immunoblotting.

Possible causes

1. Lysis may be insufficient.

2. Fusion protein may be insoluble (inclusion bodies).

Solutions

4. Some his-tagged proteins may be masked on an SDS-polyacrylamide gel by a bacterial protein of approx imately the same molecular weight. Immunoblotting can identify his-tagged proteins in these cases. Run an SDS-poly acrylamide gel of induced cells as above and transfer the pro teins to a nitrocellulose or PVDF mem-brane. Detect fusion protein using Anti-His Antibody (27-4710-01).

Solutions

1. Cell disruption is evidenced by partial clearing of the suspension or may be checked by microscopic examination.

2. If insufficient protein is found in the soluble fraction following centrifugation of the lysate, it may be necessary to alter growth conditions:– Fusion protein solubility can be

dramatically increased by lowering the growth temperature during induction. Experiment with growth temperatures in the range of 20–30°C (11, 12).

– Alter the level of induction by decreasing IPTG concentration to < 0.1 mM.

Problem: Majority of protein is found in the post-lysate pellet.SDS-PAGE analysis of samples collected during the preparation of the bac terial lysate may indicate that the majority of the his-tagged protein is located in the post-lysate pellet (Protocol 1, page 11).

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Possible causes Solutions

2. Continued. – Alter the timing of induction.

– Induce for a shorter period of time.

– Induce at a higher cell density for a short period of time.

– Increase aeration. High oxygen transport can help prevent the formation of inclusion bodies (13).

It may be necessary to combine the above approaches. Exact conditions must be determined empirically for each fusion protein.

If the above techniques do not significantly improve expression of soluble fusion protein, proteins can be solubilized from inclusion bodies using common denaturants such as 4–8 M guanidinium chloride, 4–8 M urea, detergents, alkaline pH (> 9), organic solvents (14, 15),N-lauroyl-sarcosine (Sarkosyl) (16, 17). Other variables that affect solubilization include time, temperature, ionic strength, ratio of denaturant to protein and the presence of thiol reagents (14, 15). For reviews, see references 11, 14, 15, 18 and 19.

Following solubilization, proteins must be properly refolded to regain function. Denaturant can be removed by dialysis, dilution, or gel filtration to allow refolding of the protein and formation of the correct intramol ecular associations. Critical

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Possible causes

Possible causes

1. Decrease the concentration of imidazole in the binding buffer.

Possible causes

1. Increase the duration of elution.

2. Increase the concentration of imidazole in the elution buffer.

3. Add a non-ionic detergent to the elution buffer.

Solutions

2. Continued. parameters during refolding include

pH, presence of thiol reagents and the speed of denaturant removal (14, 15, 20). Once refolded, protein may be purified by ion-exchange, gel filtration or affinity chromatography.

Solutions

1. His MicroSpin columns are pre-packed with a buffer containing 20 mM imidazole. Binding to the resin can be improved in some cases by applying the sample in 1x PN. To do so, wash the column with 1x PN once before applying the sample.

Solutions

1. In some instances, overnight elution at room temperature or 4˚C is most effective.

2. Higher con centrations of imidazole (> 400 mM) may be more effective in some cases.

3. Nonspecific hydrophobic interactions may prevent solubilization and elution of fusion proteins from Chelating Sepharose Fast Flow. Addition of a non-ionic detergent can improve results. The addition of 0.1%

Problem: His-tagged protein does not bind to His MicroSpin column.

Problem: Fusion protein is eluted poorly from His MicroSpin column.

28

Possible causes Solutions

3. Continued. Triton™ X-100 or 2% N-octyl gluco side

can significantly improve elution of some his-tagged proteins.

29

9. References1. Ford, C. F. et al., Prot. Express. Purificat. 2, 95 (1991).

2. Porath, J. et al., Nature 258, 598 (1975).

3. Smith, M. C. et al., J. Biol. Chem. 263, 7211 (1988).

4. Hochuli, E. et al., Bio/Technology 6, 1321 (1988).

5. Ljungquist, C. et al., Eur. J. Biochem. 186, 563 (1989).

6. Porath, J., Prot. Express. Purificat. 3, 263 (1992).

7. Lindner, P. et al., Methods: A Companion to Meth. Enzymol. 4, 41 (1992).

8. Studier, F. W. and Moffatt, B. A., J. Mol. Biol. 189, 113 (1986).

9. Grodberg, J. and Dunn, J. J., J. Bact. 170, 1245 (1988).

10. Gallagher, S. R., in Current Protocols in Molecular Biology Vol. 2 (Ausubel, F. M. et al. eds), John Wiley & Sons, New York, p. 10.2.2 (1995).

11. Schein, C. H., Bio/Technology 7, 1141 (1989).

12. Schein, C. H. and Noteborn, M. H. M., Bio/Technology 6, 291 (1988).

13. Schein, C. H., personal communication.

14. Schein, C. H., Bio/Technology 8, 308 (1990).

15. Marston, F. A.O., Biochem J. 240, 1 (1986).

16. Gentry, D. R. and Burgess, R. R., Prot. Express. Purificat. 1, 81 (1990).

17. Kelley, R. F. and Winkler, M. E., in Genetic Engineering Vol. 12 (Setlow, J. K., ed.), Plenum Press, New York, pp. 1-19 (1990).

18. Guise, A. D. et al., Mol. Biotech. 6, 53 (1996).

19. Pigiet, V. P. and Schuster, B. J., Proc. Natl. Acad. Sci. USA 83, 7643 (1986).

30

10. Companion products available from GE HealthcareProduct Pack Size Product number

MicroPlex™ 24 Vacuum 1 unit 27-3567-01Anti-His Antibody 170 μl 27-4710-01HisTrap™ Purification Kit 1 kit 17-1880-01Chelating Sepharose Fast Flow 50 ml 17-0575-01HiTrap Chelating HP 5 x 1 ml 17-0408-01HiTrap Chelating HP 1 x 5 ml 17-0409-01

31

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