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)