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INTRODUCTION |
This protocol describes a method for removing antibodies that react with bacterially encoded proteins by passing a crude preparation of immunoglobulins through a column containing immobilized bacterial proteins.
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MATERIALS
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Reagents
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E. coli strain used as host for preparation of expression library
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Antibody preparation that is to be used for screening
This protocol works best when using an IgG fraction, prepared by chromatography of the antiserum on protein A-Sepharose.
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Cell lysis buffer
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0.1 M sodium borate (pH 8.0)
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1 M NaCl
Sterilize the cell lysis buffer using a 0.45-µm filter, and store at room temperature. Approximately 100 ml of cell lysis buffer is required per 1 liter of bacterial culture.
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Growth medium
One liter of growth medium appropriate for the E. coli strain of choice is required.
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Lysozyme
Dissolve solid lysozyme at a concentration of 10 mg/ml in 10 mM Tris-Cl (pH 8.0) immediately before use. Make sure that the pH of the Tris solution is 8.0 before dissolving the protein. Lysozyme will not work efficiently if the pH of the solution is less than 8.0.
Use a molecular biology grade of lysozyme. Add solid lysozyme to assist lysis of bacterial cells.
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NaOH (1 N)
The preparation of 10 N NaOH involves a highly exothermic reaction, which can cause breakage of glass containers. Prepare this solution with extreme care in plastic beakers. To 800 ml of H2 O, slowly add 400g of NaOH pellets, stirring continuously. As an added precaution, place the beaker on ice. When the pellets have dissolved completely, adjust the volume to 1 liter with H2 O. Store the solution in a plastic container at room temperature. Sterilization is not necessary.
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Pancreatic DNase I
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1 mg/ml Pancreatic DNase I
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50 mM NaCl
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10 mM Tris-Cl (pH 7.5)
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1 mM MgCl2
Dissolve 2 mg of crude pancreatic DNase I (Sigma or equivalent) in 1 ml of 50 mM NaCl, Tris-Cl (pH 7.5), 1 mM MgCl2 . When the DNase I is dissolved, add 1 ml of glycerol to the solution and mix by gently inverting the closed tube several times. Take care to avoid creating bubbles and foam. Store the solution in aliquots of -20°°C. Add solid DNase I to the bacterial cell lysate to digest chromosomal DNA.
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Tris-buffered Saline (TBS)
Dissolve 8 g of NaCl, 0.2 g of KCl, and 3 g of Tris base in 800 ml of distilled H2 O. Add 0.015 g of phenol red and adjust the pH to 7.4 with HCl. Add distilled H2 O to 1 liter. Dispense the solution into aliquots and sterilize them by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2 ) on liquid cycle. Store the buffer at room temperature.
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TBS containing 0.2% (w/v) sodium azide
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Triton X-100
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METHOD
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Grow a 1-liter culture of the appropriate strain of E. coli (e.g., Y1090hsdR, XL1-Blue, or DH1) to stationary phase.
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Recover the bacteria by centrifugation at 4000g (5000 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C.
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Pour off the medium, and stand the centrifuge tubes in an inverted position to allow the last traces of medium to drain away.
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Resuspend the pellet in 100 ml of Cell lysis buffer.
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Add 200 mg of lysozyme, and incubate the bacterial suspension for 20 minutes at room temperature.
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Add 1 mg of pancreatic DNase I and 200 µl of Triton X-100.
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Incubate the bacterial suspension for 1 hour at 4°C, or until the turbidity clears and the viscosity decreases.
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Centrifuge the bacterial lysate at 8000g (8200 rpm in a Sorvall SS-34 rotor) for 20 minutes at 4°C. Carefully decant the supernatant into a fresh flask.
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Adjust the pH of the supernatant to 9.0 with 1 M NaOH.
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Determine the concentration of protein in the lysate using the Lowry, Bradford, or other method of measurement.
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Chill the extract to 0°C, and then bind the bacterial proteins to cyanogen-bromide-activated Sepharose 4B or to Affi-Gel 10 according to the manufacturer's instructions.
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Before use, equilibrate the Sepharose 4B or Affi-Gel 10 resin containing conjugated E. coli proteins in TBS containing 0.2% (w/v) sodium azide.
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Use 1 ml of settled volume of resin coupled to E. coli antigen for each milligram of IgG protein to be purified by affinity chromatography. Mix the IgG and the coupled resin and incubate for 12-18 hours at room temperature on a rotating wheel device.
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Load the slurry into a chromatography column. Recover the antibody by washing the column with TBS. Collect fractions (0.2 column volume each) until the OD280 drops to zero. Pool the fractions containing antibody, and store the pool at -20°C until it is used for immunological screening.
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REFERENCES
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1. de Wet, J.R., Fukushima, H., Dewji, N.N., Wilcox, E., O'Brien, J.S., and Helinski, D.R. 1984. Chromogenic immunodetection of human serum albumin and {alpha}-L-fucosidase clones in a human hepatoma cDNA expression library. DNA 3: 437-447.[Medline]
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Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult:
Molecular Cloning: A Laboratory Manual, Third Edition , Joseph Sambrook and David W. Russell, © 2001 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p.14.28-14.30. |
