200 -- macromolecule detection

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LABORATORY 3 -- DETECTION OF PROTEINS
LAB. 2 -- DETECTION OF PROTEINS AND POLYSACCHARIDES
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. Each group should
perform at least one control.
Biuret Procedure:
1. Obtain twelve cuvettes and label them 0 - 10, Control, 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.). Note: Your "Control tube" should also contain 10
drops and your "Unknown" should already be at ten drops.
4. Add 20 drops of NaOH (2.5%) to each tube.
5. Add three drops of Biuret reagent to all tubes, except tube 1, and mix.
Procedure for use of spectrophotometer:
1. Turn the power switch on.
2. Let warm up for 10 min.
3. Set wavelength to ___ nm.
4. 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.
5. 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%.
6. Remove blank, and place sample into sample holder.
7. Record absorbance.
8. Construct a standard curve and calculate the amount of protein in your unknown sample.
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, bluishblack 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. Repeat steps 1, 2 and 3 using starch instead of protein (each drop will contain 5 g of starch).
2. Add four drops of iodine to each tube and record absorbance at ____ nm.
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