• Resuspend lyophilized protein in:

6M Urea

90 mM KCl

50mM Tris pH 8

1mM EDTA

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