An acidic solution of monosulfonic azo dye, Acid Orange 12, reacts with basic groups on protein to form an insoluble complex. The protein dye-binding capacity is genetically controlled and is essentially constant for any given protein system. The estimate of protein content is based on a colorimetric measurement of unbound dye. The dye serves as a primary standard.


Applicable to all dairy products. The dye does not bind with non-protein nitrogen. An average non-protein nitrogen is reflected in the dye-binding capacity as derived from total nitrogen measurements.

Apparatus and Reagents

UDY Model MT-DP Protein Analyzer including an UDY Colorimeter, Reagent Dye Dispensor, Electronic Balance, Blender, and a Syringe-Pipet along with standard Reagent and Reference Dye Solutions.


1. Prepare a homogenous sample, and set UDY Dairy Tester (or Colorimeter) with values as indicated in Table II and Notes below. If desired, samples can be preserved with UDY-Pol for a shelf-life of several weeks at 20C. Homogenizing is not needed for cheese or powders.

2. Set 5 ml Syringe-Pipet to deliver 2.00 ml (1.994 g H2O at 25C). Fill by pumping with rapids short strokes. This will thoroughly mix the sample and simultaneously remove all air bubbles from the Syringe-Pipet.

3. Transfer weighed sample into a tared 60 ml polyethylene Sample Bottle. Use the weight designated in Table II. Actual weight can vary by 5% of specified weight. (See Note 2).

4. Add 40.00 ml (40.37 "0.08 g) of Reagent Dye Solution. Cap and shake vigorously for 10 to 15 seconds. Verify that longer times do not make a difference in reading. (See Notes 3 and 4).

5. Adjust temperature to 25C or make correction to reading. (See Table III).

6. Place a 19 mm fiberglass filter disk in a dropper cap; then filter 25 to 30 drops into the cuvet of a preset UDY Colorimeter. Take reading; then make any weight and temperature corrections, if needed.

Note 1: During sample preparation, when samples are diluted 10-fold, the amount of diluent to use is nine times the actual sample weight. Place diluent into Blender first, then add the sample.

Note 2: When actual weight of aliquot(s) varies more than 0.01 g from that specified, multiply % Protein reading by specified weight and divide by actual weight.

Note 3: Sample volume and weight, as well as reagent dye volume and weight, may vary when other dispensing systems are used. However, the ratio of reagent dye weight to sample weight must remain the same if the specified A and CI values are used.

Note 4: Longer shaking times may be needed by some dairy powders, when not reconstituted; depending upon solubility. To verify adequate reaction time, retest using a longer shaking time.