UDY Dye Method
Principle
An acidic solution of monosulfonic azo dye, Acid Orange 12, reacts with basic groups on proteins 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.
Scope
Applicable to most materials -- including grains, oilseeds, legumes, forages, animal products, dairy products and formulated feeds and foods. Not applicable to non-protein nitrogen or random mixtures.
Apparatus and Reagents
UDY Model SSP or MSP Protein Analyzer including a UDY Colorimeter, Reactor-R-Mill or React-R-Shaker, Reagent Dye Dispensor, Electronic Balance, and Cyclone Sample Mill along with standard Reagent and Reference Dye Solutions. Model MSP is for multiple samples and uses the React-R-Shaker.
Procedure
1. Grind 30 to 40 g of material using a 0.5 mm screen to give a homogenous powdered meal.
2. Set UDY Colorimeter with values as indicated in Table I for the specific protein system desired. Other protein system values are available from UDY Corporation. (See Note 1).
3. Weigh specified amount of finely ground sample. (See Note 2.)
4. Dispense 40.00 ml (40.37 "0.08 g) of Reagent Dye Solution into React-R_Tube or 60 ml polyethylene Sample Bottle.
5. Transfer weighed sample and about 1/4 g Filter Aid, if needed, into the dispensed Reagent.
6. Shaker React-R-Tube or Sample Bottle in Reactor-R-Mill or React-R-Shaker respectively, for time specified in Table I or until completely reacted. To verify adequate reaction time, retest using a longer shaking time. (See Note 3).
7. Adjust temperature to 25C or make a correction to reading. (See Table III).
8. Place a fiberglass filter disk in the filter cap, then filter into the cuvet of preset UDY Colorimeter. Take reading and make temperature correction as needed.
NOTE 1: Regression equations for other protein systems can be established by plotting Kjeldahl protein, P, vs equilibrium dye concn, C. (C = CI - P/A, where CI is the intercept and A is the reciprocal slope). Twenty or more representative samples covering a wide range of protein content should be tested. Use a fixed sample size such that the mid-range gives an equilibrium dye concn of about 0.70 mg/ml.
NOTE 2: When actual weight of sample varies more than 3 mg per 1000 mg (0.3%) from that specified, multiply % protein reading by specified weight and divide by actual weight.
NOTE 3: Total shaking time should not exceed 2 minutes in high speed Reactor-R-Mill or React-R-Shaker. Determine complete reaction and equilibrium dye concentrations for dye-binding capacity (DBC) value with Sample Bottles in React-R-Shaker. Adjust React-R-Tube shaking, etc. to agree. Filter immediately after reacting in the Reactor-R-Mill or React-R-Shaker.