TITLE: Cross-linking of IgG to Protein A or G Beads (S1425/S1430)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
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 20 µg or isolation of 20 µg.

[GUIDELINES]
Overview
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.

[STEPS]
SECTION: IgG Immobilization
1. 
 

Vortex and thoroughly resuspend Protein A Magnetic Beads.
SECTION: IgG Immobilization
2. Aliquot 100 µL to a sterile microcentrifuge tube.
SECTION: IgG Immobilization
3. Add 500 µL and vortex to resuspend. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 1/2)
SECTION: IgG Immobilization
4. Repeat wash: Add 500 µL and vortex to resuspend. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 2/2)
SECTION: IgG Immobilization
5. Add to the beads 80 µL.
SECTION: IgG Immobilization
6. Add 15 µL-25 µL OR 20 µg in a maximum volume of 30 µL.
SECTION: IgG Immobilization
7. Mix thoroughly and incubate at 4 °C with agitation for 30 min.
SECTION: IgG Immobilization
8. Apply magnet and remove supernatant.
SECTION: IgG Immobilization
9. Add 500 µL and vortex to resuspend. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 1/3)
SECTION: IgG Immobilization
10. Add 500 µL and vortex to resuspend. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 2/3)
SECTION: IgG Immobilization
11. Add 500 µL and vortex to resuspend. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 3/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
12. Add 1 mL to the Protein A/G immobilized antibody.(Wash 1/2) 
 
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
13. Vortex to resuspend. (Wash 1/2)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
14. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 1/2) 
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
15. Add 1 mL to the Protein A/G immobilized antibody.(Wash 2/2) 
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
16. Vortex to resuspend. (Wash 2/2)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
17. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 2/2) 
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
18. Resuspend in 1 mL containing 25 Milimolar (mM).
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
19. Mix thoroughly and incubate at 4 Room temperature for 45 min with agitation.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
20. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
21. Add 1 mL.

SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
22. Vortex to resuspend.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
23. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
24. Add 1 mL.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
25. Vortex to resuspend.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
26. Incubate for 30 min at 4 Room temperature with agitation.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
27. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
28.  Add 1 mL. (Wash 1/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
29. Vortex to resuspend. (Wash 1/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
30. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 1/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
31.  Add 1 mL. (Wash 2/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
32. Vortex to resuspend. (Wash 2/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
33. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 2/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
34.  Add 1 mL. (Wash 3/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
35. Vortex to resuspend. (Wash 3/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
36. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (Wash 3/3)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
37. Add 1 mL.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
38. Vortex to resuspend.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
39. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant.
 
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
46. Resuspend and store beads in 100 µL, 0.1% Tween 20, 0.02% sodium azide.
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
40. Add 1 mL. (1/2)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
41. Vortex to resuspend. (1/2)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
42. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (1/2)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
43. Add 1 mL. (2/2)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
45. Apply magnet for 30 s, to pull beads to the side of the tube and remove supernatant. (2/2)
SECTION: IgG Cross-linking to Protein A/G Magnetic Beads
44. Vortex to resuspend. (2/2)