• Protein G Conjugation to CMRD-SPIO
    • The following protocol is used in conjunction with the CMRD-SPIO protocol
  1. Change the CMRD-SPIO buffer to 100mM HEPES pH 4.7
  • Spin down 1mL CMRD-SPIO against 2 500 ul 100 kDa MWCO membrane at 7000 rpm for 5 min
  • Add 500 ul 100 mM HEPES pH 4.7 to each tube of CMRD-SPIO, pipet up and down a few times to mix, and spin down at 7000 rpm for 5 min
  • Add a second round of 500 ul 100 mM HEPES and spin down again.
  1. Transfer CMRD-SPIO to an Eppendorf flip tube and add 5 mg each of NHS and EDC
  • Incubate for 2 hrs at room temperature or overnight at 4oC
  1. After incubation, purify CMRD-SPIO on Spehadex G25 column with 1x PBS pH 7.4
  • Desired eluent will be easy to spot as the CMRD-SPIO is dark brown
  1. Concentrate the eluent against a 15 mL 5 kDa MWCO membrane at 2500 rpm for 30 min
  2. Add 1 mg/ml of Protein G to CMRD-SPIO solution and incubate at 4oC overnight
  3. Purify CMRD-SPIO on Spehadex G25 column with 1x PBS pH 7.4
  4. Concentrate eluent against a 15 mL 5 kDa MWCO membrane at 2500 rpm for 30 min
  5. Add desired antibody in concentration of 20 ug/ml and incubate at 4oC overnight
  6. Purify CMRD-SPIO against using a Sephadex G200 column to remove unbound antibody
  7. Store at 4oC

Edited By Mitchell Weiser 3/16/07