* **Protein G Conjugation to CMRD-SPIO**
    * // The following protocol is used in conjunction with the CMRD-SPIO protocol// 
    
    - 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.
    - 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 4<sup>o</sup>C
    - 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
    - Concentrate the eluent against a 15 mL 5 kDa MWCO membrane at 2500 rpm for 30 min
    - Add 1 mg/ml of Protein G to CMRD-SPIO solution and incubate at 4<sup>o</sup>C overnight
    - Purify CMRD-SPIO on Spehadex G25 column with 1x PBS pH 7.4
    - Concentrate eluent against a 15 mL 5 kDa MWCO membrane at 2500 rpm for 30 min
    - Add desired antibody in concentration of 20 ug/ml and incubate at 4<sup>o</sup>C overnight
    - Purify CMRD-SPIO against using a Sephadex G200 column to remove unbound antibody
    - Store at 4<sup>o</sup>C 

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Edited By Mitchell Weiser 3/16/07