Cross - linking of IgG to Protein A or G Beads ( S1425 / S1430 )
Vortex and thoroughly resuspend Protein A Magnetic Beads .
Aliquot 100 μl of bead suspension to a sterile microcentrifuge tube ( wash # 1 ) .
Add 500 μl 0.1 M NaPhosphate Buffer ( pH 8.0 ) .
( wash # 1 ) Vortex to resuspend .
( wash # 1 ) Apply magnet for 30 seconds , to pull beads to the side of the tube .
( wash # 1 ) Remove supernatant .
( wash # 2 ) Add 500 μl 0.1 M NaPhosphate Buffer ( pH 8.0 ) .
( wash # 2 ) Vortex to resuspend .
( wash # 2 ) Apply magnet for 30 seconds , to pull beads to the side of the tube .
( wash # 2 ) Remove supernatant .
Add to the beads 80 μl of 0.1 M NaPhosphate Buffer ( pH 8.0 ) .
Add 15 - 25 μl of serum OR 20 μg purified IgG in a maximum volume of 30 μl .
Mix thoroughly and incubate at 4°C with agitation for 30 minutes .
Apply magnet and remove supernatant .
( wash # 1 ) Add 500 μl 0.1 M NaPhosphate Buffer ( pH 8.0 ) .
( wash # 1 ) Vortex to resuspend .
( wash # 1 ) Apply magnet for 30 seconds , to pull beads to the side of the tube .
( wash # 1 ) Remove supernatant .
( wash # 2 ) Add 500 μl 0.1 M NaPhosphate Buffer ( pH 8.0 ) .
( wash # 2 ) Vortex to resuspend .
( wash # 2 ) Apply magnet for 30 seconds , to pull beads to the side of the tube .
( wash # 2 ) Remove supernatant .
( wash # 3 ) Add 500 μl 0.1 M NaPhosphate Buffer ( pH 8.0 ) .
( wash # 3 ) Vortex to resuspend .
( wash # 3 ) Apply magnet for 30 seconds , to pull beads to the side of the tube ( wash # 3 ) Remove supernatant .
Add 1 ml of Cross - linking Buffer ( 0.2 M triethanolamine , [ pH 8.2 ] ) to the Protein A / G immobilized antibody .
( wash a ) Vortex to resuspend .
( wash a ) Apply magnet for 30 seconds , to pull beads to the side of the tube .
( wash a ) Remove supernatant .
( wash a ) Add 1 ml of Cross - linking Buffer ( 0.2 M triethanolamine , [ pH 8.2 ] ) to the Protein A / G immobilized antibody .
( wash b ) Vortex to resuspend .
( wash b ) Apply magnet for 30 seconds , to pull beads to the side of the tube .
( wash b ) Remove supernatant .
( wash b ) Resuspend in 1 ml Cross - linking Buffer containing 25 mM DMP ( 6.5 mg DMP / ml of buffer ) .
Mix thoroughly and incubate at room temperature for 45 minutes with agitation .
Apply magnet for 30 seconds , to pull beads to the side of the tube .
Remove supernatant .
Add 1 ml Blocking Buffer ( 0.1 M ethanolamine , [ pH 8.2 ] ) .
Vortex to resuspend .
Apply magnet for 30 seconds , to pull beads to the side of the tube .
Remove supernatant .
Add 1 ml of Blocking Buffer Vortex to resuspend .
Incubate for 1 hour at room temperature with agitation .
Apply magnet for 30 seconds , to pull beads to the side of the tube .
Remove supernatant .
( wash # 1 ) Add 1 ml of PBS .
( wash # 1 ) Vortex to resuspend .
( wash # 1 ) Apply magnet for 30 seconds , to pull beads to the side of the tube .
( wash # 1 ) Remove supernatant .
( wash # 2 ) Add 1 ml of PBS .
( wash # 2 ) Vortex to resuspend .
( wash # 2 ) Apply magnet for 30 seconds , to pull beads to the side of the tube ( wash # 2 ) Remove supernatant .
( wash # 3 ) Add 1 ml of PBS .
( wash # 3 ) Vortex to resuspend .
( wash # 3 ) Apply magnet for 30 seconds , to pull beads to the side of the tube .
( wash # 3 ) Remove supernatant .
Add 1 ml Elution Buffer ( 0.1 M glycine - HCl [ pH 2.5 ] ) .
Vortex to resuspend .
Apply magnet for 30 seconds , to pull beads to the side of the tube .
Remove supernatant .
Resuspend and store beads in 100 μl PBS , 0.1 % Tween 20 , 0.02 % sodium azide
