User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2011/10/18

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Objective

Make a labeled cysteine fluorophore.

Description

  1. Measure out 5mg of 5-(Iodoacetamido)fluorescein and put in a 50mL Falcon tube.
  2. Add 19.6μL of 118mg/mL cysteine to the 50mL Falcon tube.
  3. Add 35mL of 50mM Tris, pH 7.5.
  4. Add 100μL of 2.25M DTT after about two hours of the reaction occurring at room temperature.

Notes

  • Abs at 490nm = 1.144AU for 20x dilute
  • A=bcε, b=1cm, ε=42000M-1cm-1
    • c=27.24μM for 20x dilute
    • actual concentration is 544.76μM
    • concentration was supposed to be 277.231μM (.005g/(515.3g/mol)/(.035L)*106)
  • Concentration of cysteine = ((118g/L)*(1.96*10-5L))/(121.16 g/mol)/(.035L)*106 = 545.395μM



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