Sodium Chloride → Sodium Chloride

Hybridoma supernatant (100-500 ml) was supplemented with 200 mM Tris/HCl buffer (pH 7.0) and solid NaCl to give final concentrations of 20 mM Tris and 3M NaCl, respectively. The supernatant was then clarified by centrifugation at 5500×g for 10 minutes. A 1 ml protein G affinity chromatography column (Protein G Sepharose Fast Flow, Amersham-Pharmacia) was washed with 15 ml 20 mM Tris/Cl pH 7.0 and afterwards equilibrated with 10 ml of 20 mM Tris/Cl buffer pH 7.0 containing 3M NaCl. The hybridoma supernatant containing 3M NaCl was then loaded onto the column by gravity. The column was washed with 15 ml of 20 mM Tris/Cl buffer, pH 7.0, containing 3M NaCl. Bound IgG was further eluted with 12 ml glycine/HCl buffer pH 2.8 and 1 ml fractions were collected. 100 μl of 1M Tris pH 9.0 were added to each fraction for neutralization. Fractions containing the IgG were identified by mixing 50 μl with 150 μl of a staining solution (BioRad concentrate, 1:5 diluted with water) in wells of a microplate. Positive fractions were pooled, concentrated to 1 ml in an ultrafiltration concentrator device (Centricon Plus 20, Amicon) according to the manufacturer. The concentrate was diluted with. 19 ml TBS (20 mM Tris/Cl buffer pH 7.0 containing 150 mM NaCl) and again concentrated to 1 ml. The diluting-concentrating step was repeated for two more times in order to bring IgG into TBS.

ORD record

ord-b242faa4d1744e1dba582c772be88045

Source dataset

uspto-grants-2007_10

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US07279161B2