Crosslinking of IgG to protein A sepharose
1. Take 1 ml (50 % slurry) Protein A sepharose (Pharmacia) and equilibrate in
Buffer A [50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1 % NP-40]
2. Add each antibody to beads and incubate overnight with mixing at 4 ˚C
3. Wash 3x with Buffer A containing 0.5 % NP-40
4. Wash 2x with Borate buffer pH 9.0 (see below how to make it up)
5. Add 10 ml Borate buffer pH 9.0 containing 40 mM dimethylpimelimidate
(check pH of solution)
6. Incubate with mixing for 2 hrs at room temperature to perform crosslinking
7. Wash with 40 mM ethanolamine in 0.1 M Borate buffer pH 8.0
8. Wash thoroughly as follows to make sure you get rid of all IgG that is not
covalently bound:
Buffer E (2x)
100 mM Glycine pH 3.0 (2x)
Buffer D
Buffer C
Buffer B (2x)
9. Store in 0.1 M Borate pH 8.0 + sodium azide (to prevent bacterial growth)
Borate buffers:
Solution A: 0.2 M Boric acid
Solution B: 0.05 M Sodium tetraborate
Borate buffer pH 8.0: 175 ml Solution A + 75 ml Solution B + 250 ml dH2O
Borate buffer pH 9.0: 50 ml Solution A + 200 ml Solution B + 250 ml dH2O
40 mM dimethylpimelimidate dihydrochloride: 0.1037 g / 10 ml (once you open
container, it can only be used once!)
40 mM ethanolamine: 24.4 µl/ 10 ml
Buffer B [50 mM Tris pH 8.0, 120 mM NaCl, 0.5 % NP-40]
Buffer C [50 mM Hepes, pH 7.8, 0.5 M Sodium Acetate, 10 % glycerol]
Buffer D [50 mM Hepes pH 9.0, 10 % glycerol]
Buffer E [100 mM Triethylamine in dH2O, 350 µl Triethylamine in 25 ml water]