LAB 3 -- DETECTION OF MACROMOLECULES
PROTEIN DETECTION
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.
Biuret Procedure:
1. Obtain 4 test tubes and label the "known", "no protein", "no Biuret" and "starch".
2. Add 10 drops of protein to the tube labeled "known", 10 drops of water each to the tubes
labeled "no protein" and "no Biuret", and 10 drops of starch into the tube labeled "starch".
3. Add 20 drops of NaOH (2.5%) to each tube. ***Caution -- NaOH is caustic***
4. Add three drops of Biuret reagent to all tubes, except the one labeled "no Biuret", and mix.
5. Record relative color intensity.
STARCH DETECTION
Background: Staining with iodine (iodine-potassium iodide, I2KI) distinguishes starch from
monosaccharides, disaccharides, other polysaccharides, and other macromolecules. The basis
for this test is that iodine reacts with coiled polymers of glucose (i.e., starch), producing a dark,
bluish-black color. Iodine does not react with mono-, and disaccharides, uncoiled carbohydrate
polymers, or other polymers, and therefore remains a yellowish-brown color. Controls are similar
to those used for protein detection.
Procedure:
1. Obtain 4 test tubes and label the "known", "no starch", "no iodine" and "protein".
2. Add 10 drops of starch to the tube labeled "known", 10 drops of water each to the tubes labeled
"no starch" and "no iodine", and 10 drops of protein into the tube labeled "protein".
3. Add 10 drops of iodine to each tube except the one labeled "no iodine"
4. Record relative color intensity.