Crosslinking Ab to PGS / PGA
Mix 1 vol of ascite (ascite concentration: about 1 µg/µl of IgG1) with 10 vol of PGS/PGA beads (1 µl of beads binds more or less 0.1 µg of antibody)
Incubate O/N at 4°C, on the wheel
Spin down beads (4,000 rpm for 5/10 min)
Keep SN to check for binding
2 washes with 10 vol of 0.2 M BORAX
Resuspend in 10 vol of 0.2 M BORAX
Keep an aliquote (corresponding to 1 µl of dried beads), for control
Add DMP directly as a powder (6 mg/mL, 20 mM final concentration)
Incubate 30 min at RT, on the wheel
Quench washing 2 times with 10 vol of 0.2 M ethanolamine (or tri-ethanolamine) pH 8.0
Incubate for 2 h with 10 vol of 0.2 M etanolammine (or tri-ethanolamine), pH 8.0, rotating
wash once in PBS
Resuspend in PBS, add 0.02% Na azide
SOLUTIONS
0.2 M BORAX, pH 9.0
use decahydrate: Sigma S9640 (MW=381.4)
19,07 g in 250 mL
warm up and stir extensively (tends to precipitate)
0.2 M ethanolamine, pH 8.0
100% is 16 M, dilute 1:80
pH of solution is around 10.5, bring to pH 8 with HCl
DMP powder (Sigma D8388)
NaN3 2% (100x)