Download 200 -- protein detection

LABORATORY 2 -- DETECTION OF PROTEINS
Background: Proteins may be detected by staining with the Biuret reagent. The Cu 2+ in the
Biuret reagent reacts with peptide bonds in proteins to form a violet color. Since free amino acids
do not have a peptide bond, they will not react with the Biuret reagent. The intensity of the blue
color may be measured using a spectrophotometer.
Controls: Controls could include a tube of protein in which no Biuret reagent is added, a tube in
which no protein is added, or tubes with substances besides proteins added. Each group should
perform at least one control.
Biuret Procedure:
1. Add ten drops of protein solution to a tube.
2. Add 20 drops of NaOH (2.5%) to the tube.
3. Add three drops of Biuret reagent to the tube and mix.
Experiment 1 – Detecting proteins:
1. Obtain 4 tubes and label starch, amino acid, casein, and albumen.
2. Add ten drops of each substance to each tube.
3. Perform the Biuret Procedure.
Experiment 2 – Quantifying proteins:
1. Obtain eleven cuvettes and label them 0 – 10 and Unknown.
2. Each tube labeled 0 - 10 will receive a total of 10 drops total of water and / or protein. Add 0
drops of protein to tube "0", 1 drop to tube "1", 2 drops to tube "3", etc., until you add 10 drops of
protein to tube 10. Note, each drop of protein contains 5 g of albumen.
3. Add water so each tube will contain a total of 10 drops (i.e., add 10 drops of water to tube "0", 9
drops to tube "1", 8 drops to tube "3", ect.).
4. Perform the Biuret Procedure.
5. Record absorbance using the following procedure.
a) Turn the power switch on.
b) Let warm up for 10 min.
c) Set wavelength to ___ nm.
d) Turn power switch/dark current control to adjust transmittance scale to zero. When there is
no sample in the spectrophotometer, the occluder falls into the light path, and thus no light
reaches the photodetector.
e) Place the cuvette full of sterile media (this is called a blank) into the sample holder and adjust
the absorbance control know so that transmittance reads 100%.
f) Remove blank, and place sample into sample holder.
g) Record absorbance.
h) Construct a standard curve and calculate the amount of protein in your unknown sample.