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Comparative Physiology @ Guelph

Labelling TnC with IAANS - 2008

  • Resuspend lyophilized protein in:

6M Urea

90 mM KCl

50mM Tris pH 8


50mM BME or 1 mM DTT

pH 7.5


  • Dialyze overnight into more buffer.
  • Next morning dialyze into buffer without DTT/BME

IAANS = 2-(4′-Iodoacetamidoanilo)napthalene-6-sulfonic acid

purchase from Invitrogen (Carlsbad, CA)


  • Mix probe and protein solution
  • Mixing ratio = 5X molar excess of probe to protein (5 M probe : 1 M protien)
  • Incubate, while gently shaking, at room temperature for 8 hours (in the dark) in the cold room
  • Once incubation is complete add in 2 mM DTT to stop reation


  • Dialyze 3X against:

10 mM MOPS,

90 mM KCl,

pH 7.0.

(or into complexing buffer)

  • Measure efficiency of labeling by using the Bradford assay for protein and the spec for the probe. Â Use an extinction coefficient of 24,900 Mcm at 325nm for IAANS
  • Once labeled can be frozen
  • Upper concentration of getting a labeled complex into solution is ~ 1 mg/ml