SSPBIU01.001 Page 1 of 2
Revised: 05/16/95
PROTEIN DETERMINATION
Biuret Method
PRINCIPLE:
Copper + Protein Alkaline pH > Copper-protein complexes
CONDITIONS: T = 25°C, A540nm, Light path = 1 cm
METHOD: Colorimetric
REAGENTS:
A. 0.85% (w/v) Sodium Chloride Solution (NaCl)
(Use Sodium Chloride Solution, 0.85%, Sigma Stock No. 430AG-4 or prepare 100 ml in
deionized water using Sodium Chloride, Sigma Prod. No. S-9625.)
B. 0.5% (w/v) Working Protein Standard (WPS)
(Use WPS prepared per Sigma "Preparation of Working Protein Standard for use in Protein
Determination Procedure." (See attached procedure)
C. Biuret Reagent (Biuret)
(Use Total Protein Reagent, Sigma Stock No. 541-2.)
D. Protein Sample Solution (Protein)
(Prepare a solution containing 0.5 - 4 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) ---- 0.96 0.90 0.80 0.40 ---- 1.00
Reagent B (WPS) ---- 0.04 0.10 0.20 0.60 1.00 ----
Reagent D (Protein) 1.00 ---- ---- ---- ---- ---- ----
Mix by swirling. Then add:
Reagent C (Biuret) 4.00 4.00 4.00 4.00 4.00 4.00 4.00
Mix thoroughly by vortexing and incubate for 30 minutes at 25°C. Transfer to suitable cuvettes and
record the absorbance at 540 nm for Test, Standards, and Blank.
