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Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103 |
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This page has been accessed
4 Nov 1992, rvsd 12 Oct 94, 17 Sept 95, 23 Sept 96, 15 Oct 96, 12 Oct
99, 11 Oct 00, 9 Oct 01.
From DBF's Hopkins Notebooks, III, p. 102 & VI, p. 75. |
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Microbiuret reagent is an alkaline solution of copper ions which complex with the peptide bonds in protein to produce a blue-purple color (absorption max = 310 nm) . Following Beer's Law, the color intensity of a small amount of protein mixed with this reagent should be proportional to protein concentration. Thus, this reagent can be used to assay soluble protein in unknown solutions. To do this, a "standard curve " must be generated by assaying known amounts of protein . That is the purpose of this first exercise in this series.
Wear safety goggles and handle the microbiuret reagent with care since it is a dangerous caustic solution of 35% NaOH (lye). Any hint of slipperiness on your fingers should be rinsed off with a solution of 5% boric acid (in squirt bottles) followed by thorough hand washing.
FOR A TABLE OF TWO STUDENTS
(perform experiment in pairs):
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EQUIPMENT |
SUPPLIES |
| 3 test
tube racks: 2 for 13x100 mm, 1 for 16x150 tubes 12 13 x 100 mm test tubes 2 16x150 test tubes 1 5 mL pipet for water (in 16x150 test tube) 1 2 mL pipet for protein solution (in 16x150 test tube) pipetman pipet bulb vortex safety glasses spectrophotometer, warmed up two cuvettes in test tube rack |
8 mL
microbiuret reagent
2 in Eppendorf pipettor or in 1 mL repipet (front of room) 30-40 mL dH 2O in 125 mL flask mL of mg/mL protein standard 3 in 13x100 test tube Kimwipes, paper towel |
STANDARDIZATION OF MICROBIURET
REAGENT: Here is the table for this
standardization:
4) Vortex to mix well,
let sit 15 min Wash work areas well when
finished to clean up any spilled caustic materials.
1 Our Spectronic
20s cannot measure in the UV range, but only measure absorbency down to
325 nm. 2 MICROBIURET
REAGENT: (Safety glasses should be worn during this experiment since
this solution is close to a 20% solution of NaOH. Handle with extreme
caution .)
1) Write out an experiment
table in your book (see Sample Layout of an Experiment)
2) Then set up 13x100 mm tubes for the standardization
3) Then add in sequence, accoding the to volumes in the following
table
distilled
water
mg/mL BSA
microbiuret
reagent
protein/tube
Here are the steps illustrated for setting up the standardization:
b) add the appropriate volume of protein.
c) add microbiuret reagent using a repeating pipetter.
5) Read tubes: use tube B as the blank, and
read A325
in a spectrophotometer. Read at 310 nm if you have a UV capabilities.
6) Calculate the mg protein (or in each tube. (1 g = 10
3 mg = 10
6 ug).
7) Plot standardization curve (protein vs A
325) . Here is a
typical standardization curve
.
8) Determine conversion factor to convert from optical density
at A 325
(OD) to mg protein:
Determine the slope of the line
where the curve is linear. The slope will be approximately
1.25 mg/OD unit
at A 325
).
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Solution A: 40 g NaOH (caution, caustic)100 mL dH2O to dissolve NaOH with caution |
Solution B: 400 mg CuSO4 40 mL water, agitate to dissolve 1) Q.s. To 150 mL with dH2O 2) Add solution B slowly to solution A with stirring. Store in labeled bottle marked CAUTION : caustic. |
