Protein assay

SAMPLE PREPARATION 1
Weigh 20gm of fish flesh
Macerate into smaller size
Blend in Phosphate Buffer saline at
1:10 ratio
Incubate for 24 hours at 4C in 100
rpm shaker

SAMPLE PREPARATION 2
Filter sample
Centrifuge at 4C, 14000 rpm, 15
minutes
Collect supernatant
Filter again using Whatman filter No
1
Measure the protein content against
gelatine standard using Lowry or
Bradford method

This exercise introduces students to methods of determining protein

concentrations: absorbance at 540 nm for the Biuret and 750 nm for the
Lowry assays.
Prepared solutions of gelatin at 1, 2, 3, 4, 5, and 6 mg/mL in water for Biuret
assay.
Prepare 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/L for Lowry method.
Procedure:
Biuret assay:

Mix 0.50 mL of protein with 2.50 mL of Biuret reagent. Measure the

absorbance at 540 nm after 10 minutes.

Biuret reagent:
Add, with stirring, 300 mL of 10% (w/v) NaOH to 500 mL of a solution
containing 0.3% copper sulfate pentahydrate and 1.2% sodium potassium
tartarate, then dilute to one liter.
The reagent is stable for a few months but not a year.
Adding one gram of potassium iodide per liter and storing in the dark makes
it last indefinitely.

Lowry assay:
Mix 0.25 mL of protein with 2.5 mL of Lowry reagent 1. After 10
minutes, add 0.25 mL of Lowry reagent 2 and mix well immediately.
After 30 minutes, measure the absorbance at 750 nm. Plot the
standard curve.
Lowry reagent 1: Mix one volume of reagent B (0.5% copper sulfate
pentahydrate, 1% sodium or potassium tartrate) with 50 volumes of
reagent A (2% sodium carbonate, 0.4% NaOH). Both reagents A and B
are supposed to be stable for a long time but I have had a problem
with precipitation in reagent B that seems to remedied by adding a
little NaOH.
Lowry reagent 2: Dilute commercial Folin-Ciocalteu phenol reagent
with an equal volume of water. Stable for a few days or weeks.
All series should include a zero protein (water) tube (reagent blank).

Test Sample:
Test sample can be sample with protein. Be creative in setting an
experiment.
Substitute the protein in both tests with a test sample.