Removal of cross-reactive antibody experimental steps - Database & Sql Blog Articles

The cross-reactive antibody was removed. The cell lysis buffer was 0.1 mol/L sodium acetate 1 mol/L NaCl. The cell lysis buffer was filtered through a 0.45 um filter and stored at room temperature. Approximately 100 ml of cell lysis buffer is required per 1 L of cultured bacteria. Molecular biology grade lysozyme was used for NaOH (1 mol/L) Tris-buffered saline solution (TBS) and TBS Triton X-100 enzyme and buffer lysozyme containing 0.2% (m/V) sodium azide. Solid chymase is added to aid in lysing the bacteria. Pancreatic DNase I is added to the cell lysate by the addition of solid DNase I to digest chromosomal DNA. Antibody Preparation Antibodies for library screening utilize IgG fragments prepared by protein A-Sepharose affinity chromatography. This protocol achieves the best results. For affinity chromatography with the protein Aipharoae medium, see the Harlow and Lane methods (1999). One liter of the appropriate E. coli broth is required for the medium culture. Centrifugal and rotor Sorval GSA rotors or equivalent rotors Sorval SS-34 rotors or equivalent rotor-specific equipment affinity columns 5 ml Styrofoam plastic syringes or Bi-Rad Poly-Prep columns are suitable. Hydrogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech) or Affi-GellO (Bia Rad) vector and strain Escherichia coli as a host strain for preparing expression libraries 1. Appropriate strains (such as Y1090 hsdR, XLl-Blue or DH1) The 1 L culture was cultured to a stationary phase. 2. The cells were harvested by centrifugation at 4000 g for 20 min at 4 °C with a Sorvall GSA rotor (5000 r/min). 3. Discard the medium and invert the centrifuge tube to drain the remaining medium. 4. Resuspend the pellet in 100 ml of cell lysis buffer. 5. Add 200 mg of lysozyme and incubate the broth for 20 min at room temperature. 6. Add 1 mg trypsin and 200 ul Triton X-100. 7. Incubate the broth at 4 °C for 1 h, or incubate until the supernatant becomes clear and the viscosity is reduced. 8. Centrifuge the bacterial lysate at 8000 g (8200 r/min with Sorvall SS-34 rotor) for 20 min at 4 °C and carefully pour the supernatant into another beaker. 9. Adjust the pH of the supernatant to 9.0 with 1 mol/L NaOH. 10. Detect the concentration of protein in the lysate using Lowry, Bradford or other methods. 11. Quench the extract to 0 ° C and combine the bacterial proteins with hydrogen bromide-activated Sepharose 4B or Affi-Gel 10 according to the instructions. 12. Before use, use a 0.2% (m/V) stack. The TBS of sodium nitride is equilibrated with Sepharose 4B or Affi-Gel 10 resin in combination with E. coli extract. 13. Each 1 mg IgG purified by affinity chromatography requires 1 ml of a fixed volume of resin coupled to the E. coli antigen. After mixing the IgG and the coupled resin, incubate for 12 to 18 hours at room temperature in a rotating drum. 14. Place the homogenate onto the affinity column. The antibody was eluted with TBS. The eluent (0.2 bed volume per tube) was collected until the OD280 was reduced to zero. Each tube antibody was pooled and the affinity purified antibody was stored at -20 °C for use in immunoscreening. Removal of cross-reactive antibody experimental steps