Keating:Cysteine conjugation

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Protocol for Cysteine Conjugation

this protocol is specific for the molecules to which I conjugate cysteines, but could be applied to any molecule

protein

  • resuspend lyophilized protein from NiNTA elution in water, measure concentration via Edelhoch, aliquot to 100 nmol in tubes, and lyophilize
  • resuspend a 100 nmol aliquot in 1 ml 1xTBS (50 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA) + 5 M GuHCl for 100 μM
  • reduce protein by adding 2 μl of 500 mM TCEP to each sample for 1 mM
  • heat 5 min, incubate RT 1 hr

for conjugation with iodoacetamide (IA)

  • IA - VWR 80055-298, 185 g/mol
  • want 20-200 μmol IA for 100 nmol of each protein
  • resuspend enough IA for number of samples in 1x TBS + 5M GuHCl to 1M
  • add 2.4-240 μl IA to each protein for final conc. of 20-200 mM
  • incubate 2 hr RT rocking
  • purify by HPLC

for conjugation with fluorophores

  • fluorescein maleimide (fluor/F) - Invitrogen F150, 427.37 g/mol
    • want 1μmol fluor for 100 nmol of each protein
    • resuspend enough fluor for number of samples in DMF for 20 mM
    • add 50 μl of fluor to each protein for final conc. of 1 mM
  • Alexa Fluor 568 maleimide (Alexa/A) - Invitrogen A20341, 880.92 g/mol
    • want 0.5 μmol Alexa for 50 nmol of each protein
    • resuspend 1 tube (1mg) in 113.5 ul DMSO for 10 mM (good for two proteins)
    • add 50 μl of Alexa to each protein for final conc. of 1 mM
  • incubate 4 hr RT rocking
  • use Pierce Zeba 5 ml desalting column (cat # 89892) to remove unreacted fluorophore
    • rinse columns 2x with 2.5 ml 1x TBS + 5 M GuHCl
    • add each sample to a separate column
    • rinse tubes with 100 μl (Alexa) or 200 μl (fluor) 1x TBS + 5 M GuHCl and add to columns
  • determine concentration of samples at 280 and 490 nm (fluor) or 575 nm (Alexa) by measuring spectrum from 650 or 520-240
  • take out 5 μl to run on a gel and 10 μl to run on analytical HPLC
  • aliquot in 5 tubes of 10-20 nmol each according to fluorophore concentration and rest in another tube and freeze at -80°C
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