Protein Assay by Microbiuret: Standardization

From DBF’s Hopkins Notebooks, III, p. 102 & VI, p. 75.

Adding microbiuret

tubes ready to read

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):

EQUIPMENT
3 test tube racks: 2 for 13×100 mm, 1 for 16×150 tubes
12 13 x 100 mm test tubes
2 16×150 test tubes
1 5 mL pipet for water (in 16×150 test tube)
1 2 mL pipet for protein solution (in 16×150 test tube)
pipetman pipet bulb
vortex
safety glasses
spectrophotometer, warmed up
two cuvettes in test tube rack

SUPPLIES
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 13×100 test tube
Kimwipes, paper towel

STANDARDIZATION OF MICROBIURET REAGENT:
1)  Write out an experiment table in your book  (see Sample Layout of an Experiment)
2)  Then set up 13×100 mm tubes for the standardization
3)  Then add in sequence, accoding the to volumes in the following table

Here is the table for this standardization:

a) add water first (Always add the least expensive reagents first unless there is a compelling reason to do otherwise)

b) add the appropriate volume of protein.

Adding microbiuret

c) add microbiuret reagent using a repeating pipetter.
Here is a picture illustrating the use of the Beckman Repeater

Here are the steps illustrated for setting up the standardization:

4)  Vortex to mix well, let sit 15 min
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).

microbiuret standardization curve

microbiuret standardization data

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 ).

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.)

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

3 STANDARD PROTEIN, mg/mL: Prepare 1 mg/mL solution of standard protein (bovine serum albumin [BSA], or egg albumin) by adding 100.0 mg of powder to 80 mL dH₂ O, thoroughly dissolve with stirring, avoiding foaming which denatures protein. If possible, let sit at 4C for a week to completely dissolve. Q.s. to 100.0 mL. Store at 4C. (Need ~7 mL/student).

For determination of  protein in unknowns, see:

SAMPLE LAYOUT OF AN EXPERIMENT: