1. Immunoaffinity Purifications Of Protein Complexes
  2. Conjugation of Magnetic Beads

Immunoaffinity Purifications Of Protein Complexes

Conjugation of Magnetic Beads

Necessary reagents and instrumentation

REAGENTS
  • M-270 Epoxy Dynabeads (Dynal, NY, USA)*
  • IgG or high affinity purified antibodies
  • 0.1 M sodium phosphate buffer, pH 7.4
  • 3 M ammonium sulfate
  • 100 mM Glycine HCl, pH 2.5
  • 10 mM Tris, pH 8.8
  • freshly prepared 100mM Triethylamine solution
  • PBS
  • PBS, 0.5 % TritonX-100
  • PBS, 0.02 % NaN3
  • rotor at 30 °C
  • magnet holder for eppendorf tubes
  • Tomy shaker or other type of shaker

* The amounts described in this protocol have been optimized for the use of M-270 Epoxy Dynabeads. However, this protocol was also successfully used with beads of smaller diameter. In this case, the amounts of antibody used for the conjugation will have to be changed according to the increased bead capacity of binding.

Protocol

INFO

Best if carried out in the afternoon (~ 4 pm), then washed the next morning.

  1. Weigh the necessary amount of magnetic beads in a round-bottom eppendorf tube.

  2. Wash beads with 1 mL sodium phosphate buffer, vortex for 30 s, and mix for 15 min on a Tomy shaker at room temperature.

  3. Place the tube with the bead slurry next to a magnet so that the beads are drawn to the side of the tube (not to the bottom). Remove the buffer, and wash again with 1 mL sodium phosphate buffer; vortex for 30 sec and remove buffer.

  4. Resuspend beads with IgG, anti-GFP antibodies, or other antibodies that are to be used for the immunoisolations. Use 5-10 µg Ab / 1 mg beads (Saturation of 1 mg of M-270 beads is achieved with ~7-8 µg of antibody).

  5. For the conjugation, use approximately 20 µL total volume per mg beads. This total volume includes the antibody solution, the ammonium sulfate solution, and the sodium phosphate buffer. The ammonium sulfate solution should be added last. For example, to conjugate 10 mg beads, a total volume of approximately 200 µL is appropriate. For this, add the antibody solution to the beads (to achieve the concentration mentioned in step 4), then add the sodium sulfate buffer (volume previously calculated as = 200 µL – antibody solution – 66.66 µL 3M ammonium sulfate), and then add the ammonium sulfate (will be 1/3 of the volume to get the final concentration of 1 M).

  6. Conjugation is carried out overnight on a rotor at 30 °C.

  7. Next day, in the morning, place the tube next to a magnet to draw the beads to the side of the tube. Remove supernatant and wash beads sequentially with 1mL of sodium phosphate buffer, 100 mM Glycine HCl (fast wash), 10mM Tris, freshly prepared 100mM Triethylamine solution (fast wash), four washes with PBS, one 15 min wash with PBS + 0.5 % TritonX-100, and one wash with PBS.

  8. Store the beads at 4 °C in PBS, 0.02% NaN3. Beads should be used within 2-3 weeks of conjugation. After 1 month of storage, their efficiency for isolation decreases by approximately 40 %. The unconjugated beads can be stored in organic solvent (e.g. DMF) [2 mL / 300 mg] to make aliquoting easier.

Reference

I.M. Cristea, R. Williams, B.T. Chait, M.P. Rout "Fluorescent proteins as proteomic probes" Mol Cell Proteomics (2005) 4, 1933-1941.

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