Reagents Dissolve 4.0g sodium hydroxide in about 400ml of distilled water. Add 4.5g sodium potassium tartrate. Mix to di

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Reagents
Dissolve 4.0g sodium hydroxide in about 400ml of
distilled water. Add 4.5g sodium potassium tartrate. Mix to
dissolve. Then add 1.5g CuSO4·5H2O followed
by 4.5g potassium iodide. Transfer the solution into a 500ml
volumetric flask and make up to the mark with distilled water.
Store in a tightly stoppered polyethylene bottle at room
temperature (25-35°C). Stable for 6 months.
Stock bovine immunoglobulin (BIg) solution (10mg/ml):
Dissolve 0.2g BIg in 20ml distilled water.
Working Big concentration range: 0.5 – 1.25 mg/ml
Method
1. Prepare the
following test tubes and perform the method as indicated:
Table 2: Preparation of test
tubes for the biuret method
Blank
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Unknown
Distilled H2O
1.0 ml
0.5ml
0.25 mg/ml BIg
1.0 ml
0.5 mg/ml BIg
1.0 ml
0.75 mg/ml BIg
1.0 ml
1.0 mg/ml BIg
1.0 ml
1.25 mg/ml BIg
1.0 ml
Unknown protein
0.5ml
Biuret reagent
4.0ml
4.0ml
4.0ml
4.0ml
4.0ml
4.0ml
4.0ml
2. Mix thoroughly after each
addition.
3. Incubate at room
temperature for 30 minutes.
4. Read absorbance at 540nm
for all the tubes.
Calculate the protein concentration of an unknown sample (in
mg/ml) with an absorbancy value of 0.455 using the standard curve
below.
.