Affinity chromatography assay for removal of cross-reactive antibodies
Affinity chromatography assay for removal of cross-reactive antibodies
This section describes a method for removing components of crude immunoglobulin that react with bacterial-encoded proteins using a matrix that binds bacterial proteins (de Wet et al. 1984). The method is also applicable to the removal of cross-reactive antibodies using cultured cell or tissue extracts. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Affinity chromatography assay for removal of cross-reactive antibodies
Materials and Instruments
Buffers and solutions Cell lysis buffer NaOH Tris-buffered salt solution TBS Triton X-100 Lysozyme Tryptic DNAase I Preparation of antibodies for library screening E. coli culture medium Move makings For more product details, please visit Aladdin Scientific website.
Centrifugation and rotor Affinity chromatography columns Sepharose 4B activated with hydrogen bromide
Buffers and solutions
See Appendix 12 for components of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
Cell Lysis Buffer
0.1mol/L sodium acetate
1mol/L NaCl
Filter Cell Lysis Buffer through a 0.45um membrane and store at room temperature. Approximately 100 ml of Cell Lysis Buffer is required for every 1L of cultured bacteria.
NaOH (1mol/L)
NaOH (1mol/L), Tris-Buffered Salt Solution (TBS) and TBS with 0.2% (m/V) sodium azide.
Triton X-100
Enzyme and Buffer
Lysozyme
Use molecular biology grade lysozyme. Add solid lysozyme to aid in lysis of bacteria.
Tryptic DNAase I
Add Solid DNAase I to the cell lysate to digest chromosomal DNA.
Antibody
Preparation of antibodies for library screening
This protocol gives the best results using IgG fragments prepared by protein A-Sepharose affinity chromatography. For affinity chromatography with protein Aipharoae media, see Harlow and Lane method (1999).
Medium
Culture medium
1 liter of suitable E. coli culture medium is required.
Centrifugation and rotor
Sorval GSA rotor or equivalent rotor
Sorval SS-34 rotor or equivalent
Specialized equipment
Affinity chromatography columns
5 ml glass wool-filled plastic syringes or Bi-Rad Poly-Prep columns are suitable.
Sepharose 4B (Amersham Pharmacia Biotech) or Affi-GellO (Bia Rad) activated with hydrogen bromide.
Vectors and Strains
Escherichia coli as host bacteria for preparation of expression libraries
Methods
1. 1L culture of appropriate strain (e.g. Y1090 hsdR, XLl-Blue or DH1) is grown to stationary phase.
2. Harvest the organisms by centrifugation at 4000 g for 20 min at 4°C using a Sorvall GSA turntable (5000 r/min).
3. Discard the medium and invert the centrifuge tube to drain the residual culture medium.
4. Resuspend the precipitate in 100 ml of cell lysis buffer.
5. Add 200 mg of lysozyme and incubate the culture for 20 min at room temperature.
6. Add 1 mg of tryptic DNAase, and 200 ul of Triton X-100.
7. Incubate at 4°C for 1 h, or until the supernatant turns from cloudy to clear and the viscosity decreases.
8. Centrifuge the bacterial lysate at 8000 g (8200r/min with SorvallSS-34 turntable) at 4°C for 20 min and carefully pour the supernatant into another beaker.
9. The pH of the supernatant was adjusted to 9.0 with 1 mol/L NaOH.
10. Determine the protein concentration in the lysate by Lowry, Bradford or other methods.
11. Quench the extract to 0°C and bind the bacterial proteins to hydrogen bromide-activated Sepharose 4B or Affi-Gel 10 according to the instructions.
12. Equilibrate the Sepharose 4B or Affi-Gel 10 resin bound to the E. coli extract with TBS containing 0.2% (m/V) sodium azide prior to use.
13. For every 1 mg of IgG purified by affinity chromatography, 1 ml of a fixed volume of resin coupled to E. coli antigen is required. the IgG and coupled resin are mixed well and incubated in a rotary drum for 12-18 h at room temperature.
14. Load the homogenate onto an affinity chromatography column. Elute the antibody with TBS. Collect the eluate (0.2 column bed volume per tube) until the OD280 drops to 0. Combine the tubes and store the affinity-purified antibody at -20°C for use in immunoscreening.