Purification of monoclonal antibodies from hybridomas
From Genome@Yale
- Affinity columns: protein G for mouse IgG1 and protein A for mouse IgG2a, IgG2b, IgG3
- Set appropriate volume of protein G sepharose column according to the common rule: culture supernatant contain 20-50 ug/mL antibody, 1 mL of wet beads bind approximately 10-20mg antibody, wash column with 100 mM Tris-HCL pH 8.0.
- Adjust cell culture supernatant pH by adding 1/10 volume of 1.0M tris-HCL pH8.0, pass it through protein A column at speed of 2 mL/min.
- Aash column with at least 10 column volume of 100mM Tris-HCL then wash with 10mM Tris-HCL.
- Elute the column with 50 mM glycine(pH3.0), add this buffer stepwise at 1ml per time, collect elute fraction into 1.5 ml eppendorf tube containing 100ul 1M Tris-HCL for immediate neutralization of antibody solution
- During collecting elute fractions, use Bradford or BCA solution to monitor eluted protein
- Running 5 uL of each fraction on 11% SDS-PAGE gel to check the purity of antibody
- Combine the fractions containing antibody, determine the antibody concentration by measuring OD280nm (1 OD = 0.75 mg/mL)
- Make series dilution of antibody such as 1:500, 1:1,000, 1:2,000, 1:3,000, 1:4,000, perform western blot to determine the optimal titre of the antibody, when doing titration, the antigen should be Myc (for 9E10) or HA (for 12CA5) tagged protein which was confirmed by other Western bolt.
