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Method for IgG Purification

Solutions

  • PROSEP-A binding buffer A: 1.5 M Glycine/NaOH, 3 M NaCl, pH 9.0
  • PROSEP-A elution buffer B2: 0.2 M Glycine/HCl, pH 2.5
  • PROSEP-A neutralization buffer: 1 M Tris/HCl, pH 9.0

Procedure

  1. Recombinant proteins were expressed in Escherichia coli.
  2. 200 mg of PROSEP-A media were placed in Ultrafree-MC 0.45 μm filter basket.
  3. The columns were equilibrated with 400 μL of binding buffer A and centrifuged for 1 minute at 100 xg.
  4. 200 μL of rabbit serum were diluted 1:1 with binding buffer and the entire volume was loaded into the spin column containing PROSEP-A resin.
  5. Devices were placed on a shaker for 15 minutes at room temperature and centrifuged at 100 xg for 5 minutes. Flow-through was collected for future analysis.
  6. Three consecutive washes of 400 μL each were performed by adding 400 μL of binding buffer A and centrifuging at 2,000 xg for 2 minutes each.
  7. 200 μL of elution buffer B2 were added and centrifuged for 2 minutes at 2,000 xg.
  8. 26 μL of neutralization buffer were added to each collection tube. A second elution was collected after repeating the same process one more time.

Method for His-tagged C-RP Purification

Solutions

  • Lysis buffer: 50 mM sodium phosphate, 300 mM sodium chloride,10 mM immidazole, pH 7
  • Binding buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM immidazole, pH 7
  • Wash buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 20 mM immidazole, pH 7
  • Elution buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 250 mM immidazole, pH 7
  • 1 mg/mL Lysozyme stock
  • Benzonase

Procedure

  1. Cells were prepared at a 10X concentration using lysis buffer. Lysozyme was added to a concentration of 0.1 mg/mL. To reduce the viscosity, benzonase was added to the lysate. The lysates were clarified by centrifugation.
  2. 200 μL of the 50% resin slurry were added to the Ultrafree-MC device and the residual fluid was removed by centrifugation for 1 minute at 500 xg.
  3. The resin was equilibrated with 500 μL of binding buffer and centrifuged for 2 minutes at 500 xg.
  4. 500 μL of the clarified lysate were added to the resin.
  5. The His-tagged proteins were bound for 30 minutes with light agitation.
  6. The lysate was removed by centrifugation at 500 xg for 10 minutes.
  7. The resin was washed with 500 μL of wash buffer for 5 minutes with agitation. The wash solution was removed by centrifugation for 5 minutes at 500 xg. This step was repeated two more times.
  8. 250 μL of elution buffer were added to the Ultrafree MC device and mixed for 5 minutes. Purified protein was recovered by centrifugation at 500 xg for 1 minute.

Figure A: Rabbit lgG purification on PROSEP-A resin in Ultrafree-MC devices.
Figure A: Rabbit lgG purification on PROSEP-A resin in Ultrafree-MC devices.
Lane 1: Molecular weight standards
Lane 2: Rabbit serum
Lane 3: Flow through
Lanes 4 - 6: Three consecutive washes
Lanes 7, 8: Eluted lgG from two devices

Results

The results of purifying rabbit IgG using Ultrafree - MC centrifugal devices are shown in Figure A. The device was challenged with approximately 14 mg of total protein, with an estimated IgG content of 1. 5-2 mg. The original serum, flow-through, three washes and two eluted fractions were analyzed by SDS-PAGE. The total amount of purified IgG, as estimated by OD280, was 1.2 mg and 1.1 mg on two devices processed in parallel. The whole procedure was completed in less than 1 hour. This method can be useful for monitoring the titer of antigen- specific antibodies after immune activation, or whenever small amounts of IgG need to be purified.

Figure B shows the results of His-tagged protein purification in Ultrafree-MC devices using Ni-NTA agarose and BD Talon™ resin. Two recombinant proteins were purified: C-RP, highexpressing protein of 26 kDa; and RT66, low expressing protein of 66 kDa. As seen in Figure B, both purifications resulted in high purity proteins. The amount of proteins purified out of 500 μL of lysate was 32-35 μg for C-RP protein and 8 μg for RT66.

The data show that affinity batch purification can be effectively performed on a small scale using Ultrafree-MC devices loaded with resin. The process combines the high efficiency of batch binding and washing with the handling convenience of a mini-spin column. With minimal hands-on time, the method provides flexibility of resin to lysate ratio and binding conditions, independent of centrifugation speed and rotor angle. This method is applicable to recombinant protein purification, antibody purification and immunoprecipitation.

Figure B: His-tagged protein purification using Ultrafree-MC devices.
Figure B: His-tagged protein purification using Ultrafree-MC devices.
Lane 1: Molecular weight standards
Lane 2: E.coli lysate expressing C-RP protein
Lane 3: E.coli lysate expressing RT66 protein
Lanes 4, 5: Protein purified on Ni-NTA resin
Lanes 6, 7: Protein purified on BD Talon resin

Large Scale Purification

Protein A/G Chromatography

Required Equipment and Equipment

  • Protein A or G agarose gel column (10 mL or 5 mL of packed beads. See guidelines below for choice of protein A or protein G.)
  • Ice-cold Tris-buffered saline (TBS). See recipe below
  • 5% sodium azide solution
  • Neutralization Buffer (NB). See recipe.
  • Elution Buffers (pH 2.7 and pH 1.9). See recipes.
  • Centrifuge tubes and microcentrifuge tubes
  • Preparative centrifuge and microcentrifuge
  • pH strips
  • Microtiter plate reader with 600 nm filter
  • Glass Columns

Guidelines for Choosing Protein A Agarose or Protein G Agarose

Proten G bind with different affinities to Protein A and Protein G conjugated agaroses. See Protein A/G binding affinities in the Appendix to choose the best affinity agarose for your antibody.

Buffer Preparation

To prepare TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% sodium azide) add the following to 800 mL of distilled water:

  • 6. 06 g of Tris base
  • 8. 77 g of NaCl
  • 10 mL of 5% sodium azide stock solution. (Do not add if the buffer will be used in assays of cellular response or function.)

Mix well and adjust the pH to 7.4 with 5 N Hcl. Bring the volume up to 1 L with distilled water and recheck the pH after chilling on ice.

To prepare 100 mL of NB (1 M Tris-HCl, pH 8.0, 1. 5 M NaCl, 1 mM EDTA, 0.5% sodium azide), add the following to 80 mL of distilled water:

  • 12. 1 g of Tris base
  • 8. 7 g of NaCl
  • 200 μL of 0.5 M EDTA
  • 10 mL of 5% sodium azide

To prepare 100 mL of pH 2.7 Elution Buffer (50 mM Glycine-HCl, pH 2.7), add the following to 80 mL of distilled water:

  • 0. 38 g of glycine
Mix thoroughly and adjust the pH to 2.7 with 5 N Hcl. Add distilled water to a final volume of 100 mL.

To prepare 100 mL of pH 1.9 Elution Buffer (50 mM Glycine-HCl, pH 1.9), add the following to 80 mL of distilled water:

  • 0. 38 g of glycine
Mix thoroughly and adjust the pH to 1.9 with 5 N Hcl. Add distilled water to a final volume of 100 mL.

Procedure

Section I: Preparation of a Protein A Agarose or Protein G Agarose Affinity Column

Typically columns containing 5 mL or 10 mL of packed protein A/G agarose are prepared. The size of the column is determined by the binding capacity of protein A and protein G and the amount of antiserum that must be processed. Protein A and protein G bind about 20 mg of IgG per mL of conjugated agarose. Therefore the binding capacity of a 10 mL (5 mL) column is 200 mg (100 mg) of IgG. A high-titer rabbit antiserum has roughly 5 mg/mL of IgG; mouse ascites has roughly 10 mg/mL of IgG; and goat or sheep antiserum has roughly 20 mg/mL of IgG. Use the guidelines below to choose a column size to avoid exceeding the capacity of the column. Do not exceed 90% of the binding capacity of the column.

10 mL Bead Volume Protein A/G Affinity Column
Source of Antibody Concentration Vol. for Capacity Vol. for 90% Capacity
Rabbit Antiserum 5 mg/mL 40 mL 36 mL
Mouse ascites 10 mg/mL 20mL 18mL
Goat/sheep antiserum 20 mg/mL 10mL 9mL

5 mL Bead Volume Protein A/G Affinity Column
Source of Antibody Concentration Vol. for Capacity Vol. for 90% Capacity
Rabbit Antiserum 5 mg/mL 20 mL 18 mL
Mouse ascites 10 mg/mL 10mL 9mL
Goat/sheep antiserum 20 mg/mL 5mL 4.5mL

Pouring the Protein A/G Affinity Colum

  1. Use a pipette to transfer the desired volume of a 50% protein A/G agarose slurry to a vacuum flask. Transfer enough slurry to prepare a column with the desired bed volume and capacity.
  2. Place a stopper in the vacuum flask and apply vacuum for at least 15 minutes at room temperature. This step removes gas from the agarose and is necessary to prevent bubbles in the column that would reduce the column’s capacity and resolution.
  3. While degassing the agarose, chill the TBS on ice. Prepare TBS without azide for antibodies that will be used in vivo or in cellular assays. TBS is used to equilibrate and wash the protein A/G column.
  4. Slowly add the degassed protein A/G agarose to a glass column using a wide bore pipette to prevent rupturing the beads.
  5. Pack the column at a flow rate of about 1-2 mL per min. Do not let the column run dry! Using a Mariotte flask or pump may control flow rate.
  6. Wash the column with 10 bed volumes of ice-cold TBS. This step chills the column, which reduces nonspecific binding of proteins and slows the metabolism of any remaining viable microbes.

Section II. Preparation of Antiserum or Ascites for Affinity Chromatography

  • Throughout this Section, “Antiserum” Refers to Antiserum or Ascites.
  • Thaw the antiserum in ice water or the refrigerator overnight, to prevent aggregation of proteins. Any protein aggregate that forms during thawing may be dissolved by briefly warming the thawed antiserum to 37°C.
  • Add sodium azide to a final concentration of 0.05%, which is a 1:100 dilution of a 5% antiserum stock solution. Caution:Sodium azide is toxic; wear gloves and handle the stock solution with care.
  • Clarify the antiserum by centrifugation at 15,000 xg for 5 minutes at 4°C. This step is done to sediment aggregates of denatured protein and lipid; it is an important step because it removes material that can foul and block the column.
  • Remove the clarified antiserum that lies between the floating lipid and the pellet. Additional filtration may be required to remove residual lipid.

Section III. Affinity Chromatography Using Protein A/G Agarose

  1. Save a 500 μL sample of the clarified antiserum for testing along side the purified IgG because the antibody may be acid labile.
  2. Add the clarified antiserum to the column at a flow rate of 2 mL per minute. (Calibrate the flow rate using a 15 mL conical centrifuge tube as the receiving vessel. Adjust the stopcock on the column to give a flow of 1-2 mL per minute.) Pass the anti-serum through the column twice and save the flow through in case the antibody did not bind to the column.
  3. Wash the column with a volume of TBS equal to 10x the volume of antiserum loaded on the column. For example, if 30 mL of antiserum were loaded on the column, wash with 300 mL of PBS. Save the wash in case the antibody was eluted.
  4. After washing the column, collect a 20 μL fraction and test for protein by adding it to 180 μL of Coomassie Blue reagent to test for protein. If the sample turns blue, wash the column with an additional 50 mL of TBS and repeat the analysis for protein.
  5. Prepare 1.5 mL microcentrifuge tubes for the collection of eluted antibody. Plan one tube for each mL of antiserum plus five extra 1.5 mL tubes. Add 100 μL of NB to each tube. Note that IgG may be eluted from protein A/G agarose by a change in temperature, ionic strength, and/or pH. All of these conditions affect the binding between the charged amino acids of protein A/G and the Fc region of the IgG heavy chain. When eluting with acidic buffer, the acid must be neutralized as quickly as possible to prevent denaturation of the IgG.
  6. Once the column has been washed, drain the TBS from the column bed to avoid unnecessary dilution of the Elution Buffer. Do not let the column stand dry.
  7. Gently add Elution Buffer pH 2.7 at room temperature to the column. Be careful not to disrupt the column bed. Use 15 mL of Elution Buffer pH 2.7 if less than 20 mL of antiserum was loaded. Use 20 mL of Elution Buffer pH 2.7 if 20-30 mL of antiserum was loaded. Use 25 mL of Elution Buffer pH 2.7 if 30-36 mL of antiserum was loaded. Note that these volumes are guidelines and Elution Buffer must be added until all protein has been eluted from the column.
  8. Collect roughly 1 mL fractions in the tubes prepared in step 5 above. Mix each fraction immediately and place on ice before collecting the next fraction. Neutralizing the acid pH prevents denaturation of the antibodies and loss of biological activity.
  9. Add 10 mL of Elution Buffer pH 1.9 at room temperature to the column; be careful not to disrupt the column bed. Collect, mix, and save fractions as described in step 8 above.
  10. Remove a 20 μL sample of each fraction and add to 180 μL of Coomassie Blue reagent in a microtiter plate, to monitor protein elution. Continue to collect fractions until the color drops to or just below background.
  11. Read the microtiter plate at 600 nm and combine all fractions with an absorbance greater than or equal to 0.2 above background at 600 nm. For example, if the background is 0.18, combine all fractions with an absorbance greater than 0.38.
  12. Once the absorbance has dropped below 0.2, wash the column with 200 mL of ice-cold TBS with 0.05% sodium azide.
  13. Store the column at 2-8°C.
  14. Use pH paper to check the pH of the combined fractions. If the pH is less than 7.0, use NB to adjust the pH to approximately 7.4.
  15. Use the Bradford Assay with IgG as a standard, to determine the concentration of the antibody in the combined fractions. Add glycerol to 10% of the total volume, if the antibody concentration of the combined fractions is less than 0.5 mg/mL.
  16. Store the purified antibody at 2-8°C.

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