Rabbit IgG Conjugation to Dynabeads   

Abstract

Materials and Reagents

1. Invitrogen Conjulation kit (143.11D), contains C1, C2, HB, LB, SB solutions
2. IgG (Sigma-Aldrich, catalog number: I5006-10 mg )
3. Dynabeads M-270 Epoxy 300 mg (Life Technologies, Invitrogen™)
4. Phosphate buffered saline (PBS) tablet
5. 30% NaN₃ (fresh)
6. 20% Tween 20
   

Equipment

1. Hula mixer (Life Technologies, Invitrogen™) or other rocker capable of 360° rotation
2. Magnet for 15 ml tubes
3. Spectrometer
   

Procedure

Day 1

1. Dissolve rabbit IgG in cold 1x PBS to a final concentration of 10 mg/ml.
2. Centrifuge the rabbit IgG at 14,000 rpm for 10 min at 4 °C, and save the supernatant. 
3. Determine the IgG concentration using the Bradford method on a spectrometer. 
4. Add 5 ml C1 of the Conjulation Kit to the 300 mg dynabeads and mix by pipetting in the original glass vial. Then transfer to a 15 ml tube. Add C1 to 12 ml, split to another three 15 ml tubes, each tube with 3 ml beads.
5. Remove the supernatant from the tube on a magnet.
6. Add 300 μl rabbit IgG from step 1, 2.7 ml C1 to the beads, mix by pipetting.
7. Add 3 ml C2 and mix by pipetting again.
8. Wrap in aluminum foil and incubate at 37 °C for 16-24 h on Hula mixer set at 15-20 rpm.
   

Day 2

1. Centrifuge 1,000 x g, 5 min. Remove the supernatant (save supernatant for calculation of protein conjulation efficiency).
2. Wash each tube with 1,000 μl HB in each tube (add HB, then transfer to a 2 ml tube).  Combine supernatant.
3. Wash each tube with 1,000 μl LB. Combine supernatant.
4. Wash each tube with 1,000 μl SB. Combine supernatant.
5. Wash each tube with 1,000 μl SB and incubate at room temperature (RT) for 15 min.  Combine supernatant.
6. Wash each tube with 1,000 μl PBS+0.05% Tween at RT for 10 min x 3 times.
7. Resuspend beads in each tube with 2 ml SB, add NaN₃ to final concentration of 0.02%. Then split each tube of beads (400 μl) to 5 tubes containing 600 μl SB each with 0.02% NaN₃.
   

References

1. Li, X., Gianoulis, T. A., Yip, K. Y., Gerstein, M. and Snyder, M. (2010). Extensive in vivo metabolite-protein interactions revealed by large-scale systematic analyses. Cell 143(4): 639-650.