Materials
Reagents:
12 mg/mL bovine serum albumin (BSA)
Biuret reagent
Test Solutions:
water, dilute gelatin
egg albumen
unknown protein solution 2
unknown protein solution 3
unknown protein solution 4
Supplies:
test tubes
test tube racks
wax pencil or marker
1 mL pipettes
5 mL pipettes
parafilm
spectrophotometer
cuvettes
holder for cuvettes
Kimwipes
vortex
Procedure
Refer to Appendix 6 for the use of the spectrophotometer. Turn on the spectrophotometer and set the wavelength at 545 nm. Wait 15 minutes to allow the instrument to warm up before taking any readings.
Label six test tubes (numbers 1-6), making certain all test tubes are clean and dry. (To facilitate measuring, you can use capped graduated disposable tubes if available.)
To test tube 1, add 1 mL of water and 5 mL of Biuret reagent. This sample is called a blank.
To test tubes 2-6, add H2O, BSA, and Biuret reagent in that precise order, according to Table 6.4.
Prepare test tubes 7-11 for your known and unknown protein samples by adding 1 mL of sample and 5 mL of Biuret reagent, according to Table 6.5.
For uncapped tubes, cover all 11 test tubes with parafilm and mix, either by using a vortex or by inversion. If using capped disposable tubes, there is no need for parafilm.
Arrange 11 cuvettes in order. DO NOT MARK THE CUVETTES as it will interfere with proper reading by the spectrophotometer. Handle the cuvettes by the rim only. (See Appendix 6 for proper care of cuvettes.)
Pour the contents of each test tube into a separate cuvette.
Let tubes stand 5 minutes before reading in the spectrophotometer.
Before inserting into the spectrophotometer, wipe each cuvette with a Kimwipe or soft tissue to remove oils and fingerprints from the side of the tube.
TABLE 6.4 Bovine serum albumin (BSA) dilutions for the Standard Curve.
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