PURIFICATION OF RAT ANTI-MOUSE mAbs

 

Pack a column of 1mL of protein G sepharose 4 Fast Flow -Pharmacia (capacity ~7mg mAb/ml of gel bed matrix)

Wash with 10 mL PBS or saline

Pre-elute with 0.1M glycine-HCL (pH 2.7) until baseline absorbance at 280nm is obtained.

Re-equilibrate the gel bed with running buffer (20mM sodium phosphate, pH 7.2),

Run through hybridoma supernatant (100ml -1L) at a flow rate of 1ml/min.

Wash gel bed with running buffer to ensure a baseline absorbance reading of 280nm.

Elute mAb from the gel bed with 0.1M glycine-HCL (pH 2.7).

Collect in fractions of 1-2 mL until the 280nm absorbance reading returned to baseline level

Immediately neutralise to pH 7.0 by the addition of 1M Tris-HCL (pH 9.0).

Dialyse overnight against PBS at 4°C, with at least one change (or 3 30 mins in 100 fold volume change).

Measure concentration of purified mAb with a spectrophotometer at 280nm, using a value of 1.4 for the absorbance of 1mg/ml solution

Store mAbs at a concentration of 1- 2mg/ml. Test reactivity by either indirect immunofluorescence and/or flow cytometry before use.