Cross-linking of IgG to Protein A or G Beads (S1425/S1430)
New England Biolabs
Protein A
Protein G
cross-linkage
cross linked
covalent cross-linking of the IgG to protein A/G
igG purification to beads
Abstract
This protocol consists of an IgG purification step followed by covalent cross-linking of the IgG to the Protein A/G solid support. For IgG that has been previously purified, proceed directly to the cross-linking protocol.
Before start
The IgG Purification protocol is for the binding of 20µg or isolation of 20µg.
Steps
IgG Immobilization
Vortex and thoroughly resuspend Protein A Magnetic Beads.
Aliquot 100µL to a sterile microcentrifuge tube.
Add 500µL and vortex to resuspend. Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 1/2)
Repeat wash: Add 500µL and vortex to resuspend. Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 2/2)
Add to the beads 80µL.
Add 15µL-25µL OR 20µg in a maximum volume of 30µL.
Mix thoroughly and incubate at 4°C with agitation for 0h 30m 0s.
Apply magnet and remove supernatant.
Add 500µL and vortex to resuspend. Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 1/3)
Add 500µL and vortex to resuspend. Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 2/3)
Add 500µL and vortex to resuspend. Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 3/3)
IgG Cross-linking to Protein A/G Magnetic Beads
Add 1mL to the Protein A/G immobilized antibody.(Wash 1/2)
Vortex to resuspend. (Wash 1/2)
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 1/2)
Add 1mL to the Protein A/G immobilized antibody.(Wash 2/2)
Vortex to resuspend. (Wash 2/2)
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 2/2)
Resuspend in 1mL containing 25millimolar (mM).
Mix thoroughly and incubate at 4Room temperature for 0h 45m 0s with agitation.
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant.
Add 1mL.
Vortex to resuspend.
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant.
Add 1mL.
Vortex to resuspend.
Incubate for 0h 30m 0s at 4Room temperature with agitation.
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant.
Add 1mL. (Wash 1/3)
Vortex to resuspend. (Wash 1/3)
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 1/3)
Add 1mL. (Wash 2/3)
Vortex to resuspend. (Wash 2/3)
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 2/3)
Add 1mL. (Wash 3/3)
Vortex to resuspend. (Wash 3/3)
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (Wash 3/3)
Add 1mL.
Vortex to resuspend.
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant.
Add 1mL. (1/2)
Vortex to resuspend. (1/2)
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (1/2)
Add 1mL. (2/2)
Vortex to resuspend. (2/2)
Apply magnet for 0h 0m 30s, to pull beads to the side of the tube and remove supernatant. (2/2)
Resuspend and store beads in 100µL, 0.1% Tween 20, 0.02% sodium azide.
