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SSBTCA01.001 Page^1 of^3
Biuret Method using TCA Precipitation
CONDITIONS: T = (^25) °C, A540nm, Light path = 1 cm
METHOD: Colorimetric
REAGENTS:
A. 0.85% Sodium Chloride Solution (NaCl) (Use Stock No. 430AG-4 or prepare 100 ml in deionized water using Sodium Chloride, Prod. No. S-9625.)
B. 1% Working Protein Standard (WPS) (Use WPS prepared as per Working Protein Standard Procedure.)
C. Biuret Reagent (Biu) (Use Stock No. 540-2.)
D. Trichloroacetic Acid (TCA) (Use Stock No. 490-10)
E. Protein Sample Solution (Pro) (Prepare a solution containing 1 - 3 mg protein/ml of Sample in Reagent A.)
PROCEDURE:
Pipette (in milliliters) the following reagents into suitable containers:
Test Std 1 Std 2 Std 3 Std 4 Std 5 Blank
Reagent A (NaCl) ---- 0.98 0.95 0.90 0.70 0.50 1. Reagent B (WPS) ---- 0.02 0.05 0.10 0.30 0.50 ---- Reagent E (Pro) 1.00 ---- ---- ---- ---- ---- ----
Mix. Then add:
Reagent D (TCA) 0.10 0.10 0.10 0.10 0.10 0.10 0.
SSBTCA01.001 Page^2 of^3
Biuret Method using TCA Precipitation
Mix thoroughly and place the containers in an ice bath for 10 minutes. Centrifuge for 20 minutes in a clinical centrifuge. Remove and discard the supernatant. Then add:
Test Std 1 Std 2 Std 3 Std 4 Std 5 Blank
Reagent A (NaCl) 1.00 1.00 1.00 1.00 1.00 1.00 1.
Mix thoroughly until pellet dissolves. Then add:
Reagent C (Biu) 4.00 4.00 4.00 4.00 4.00 4.00 4.
Mix thoroughly and seal the containers. Incubate for 30 minutes at (^25) °C. Transfer to suitable cuvettes and record the absorbance at 540 nm for Test, Standards, and Blank.
CALCULATIONS:
?A540nm Standard = A540nm Std - A540nm Blank
Find the slope (M) by plotting the ?A540nm Standards vs Protein concentration.
Sample Determination:
Determine the concentration of protein using the following equation:
A540nm Test - A540nm Blank mg Protein = ___________________________________ (M)
(mg Protein) (100) % Protein = ______________________ (mg solid/ml Reagent D)
For Products that are liquid:
(mg Protein) (Dilution) mg Protein/ml = ___________________________________ (ml Reagent D)
100 = Conversion to percentage
