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Cysteine Assay Protocol

Oksana’s Cysteine Assay


Solutions:

800μM Cysteine Stock with 1x10-3M EDTA (freshly prepared!)

0.1M Tris Buffer (pH 7.05) with 1x10-3M EDTA

2.5mM DTNB in 50mM Ammonium Acetate, pH 5.0, with 1x10-3M EDTA


Prepare all solutions with MilliQ water

First dissolve DTNB in Ammonia until solution clear then add rest of components

[Cys] μM

Tris Buffer (ml)

Water (μL)

DTNB (μL)

Cys (μL)

0.0

2.60

200

200

0

13.3

2.60

150

200

50

26.7

2.60

100

200

100

40.0

2.60

50

200

150

53.3

2.60

0

200

200


The total volume must be 3mL!!!!


Sample:

Prepare samples as you have done the standards, but replace Cys with your sample.

You must decide how much volume to put into mixture. If you expect you sample to be weak pipette 200μL into the mixture; however, if your sample is very concentrated you may want to put in less than 50μL.

Once standards have been prepared, measure them immediately at 412nm. Remember to measure the blank!

Leave the sample to react for 10-15 minutes before measuring!!! You could even monitor at 5, 10 and 15 minutes until the reaction is complete i.e. no change. Remember to measure the blank!!