Download 5 Quantitative Determination of Proteins

5 Quantitative Determination of Proteins
Objectives:
A) To prepare a standard curve for the Bio-Rad assay and B) to determine
and compare the unknown concentration of a solution of a protein from
this curve.
Introduction: There are many methods for determining protein concentration. Some of
these methods depend on the reactions of reagents with peptide bonds or
amino acid side chains of the protein; others depend on the binding of a
reagent (dye) to the protein.
The Biuret Method
In this method, the reagent, a dilute copper sulfate solution at alkaline
pH, reacts with peptides or proteins. This complex undergoes a blue to
violet color transformation that requires five minutes to complete. The
protein is quantitated at 540 nm. This method requires relatively large
quantities of protein (1 - 20 mg protein / mL) for detection. Additionally,
it is sensitive to a variety of nitrogen-containing substances that could be
in the protein solution, thereby increasing the likelihood of erroneous
results.
The Lowry Test
The Lowry test is the most sensitive quantitative colorimetric assay
for protein detection. This test requires only 0.005 to 0.3 mg protein per
mL for detection. It is a modification of the biuret method. The intense
blue-green color formed in the Lowry test comes from the reaction of the
phosphomolybdotungstate in the Lowry reagent with the W and Y
residues in the protein. This assay method, however, can give low values
if the protein under examination does not have a significant number of W
and Y residues.
The Bradford (Bio-Rad) Method
This method employs a dye that binds to the protein to form a blue
complex. It can detect from 0.2 to 1.4 mg of protein per mL. The
Bradford method uses the negatively charged dye Coomasie Brilliant Blue
G-250, which binds to positive chains of the protein, to give the blue
complex. The red form of this dye predominates in solution before a
protein is added. The color changes into the blue form upon complexing
to the protein – the more concentrated the protein, the more intense the
blue color. This method is more rapid and less susceptible to interfering
substances than either of the above methods. The color develops within 2
- 5 minutes and is stable up to 24 hours. It is for these reasons that it is the
most popular method of protein quantitation. Consequently, it is the
method we will use in today’s experiment.
The Protein
The protein we will analyze is bovine serum albumin (BSA).
Albumin is a serum protein that transports fatty acids and is important in
maintaining plasma pH. In protein quantitation assays, BSA serves as a
reference protein that is used to construct protein standard curves. Other
proteins can be used depending on the physical/chemical properties of
your protein of interest.
Procedures for the Bio-Rad Protein Assay:
Procedures for the Bio-Rad Protein Assay:
I.
Prepare a standard curve
You will be given a stock solution of 0.8 mg/mL bovine serum albumin
(BSA)
You will need to make 6 standards. Make 1 mL of each of the following
concentrations:
10 µg/mL
20 µg/mL
40 µg/mL
60 µg/mL
80 µg/mL
100 µg/mL
I have not given you the exact procedure to follow because you should be able to
calculate the volumes required to make these standards. Review the handout from the
first day of class.
II.
Prepare the plate
To wells A4 – A6 add 160 µL water (this is the blank)*
To wells B4 – B6 add 160 µL of 10 µg/mL standard
To wells C4 – C6 add 160 µL of 20 µg/mL standard
To wells D4 – D6 add 160 µL of 40 µg/mL standard
To wells E4 – E6 add 160 µL of 60 µg/mL standard
To wells F4 – F6 add 160 µL of 80 µg/mL standard
To wells G4 – G6 add 160 µL of 100 µg/mL standard
To wells H4 – H6 add 80 µL of the protein sample of unknown concentration and
80 µL water
Add 40 µL of Stock BioRad reagent to each well
MIX WELL WITH THE PIPETTE TIP, last year the groups that mixed well
got good results those who did not had very poor results.
WAIT AT LEAST 5 MINUTES BEFORE READING
•
III.
The plate reader will average the “blank” absorbance values and subtract this
average from every other well. You will also use this as one of the points on
your standard curve.
Read the plate
Open protocol “proteinquant#” where # is your section number
Click on Icon that reads “Read”
Fill in the information:
Section
Name
Experiment
Click on button that reads “Start Reading”
A window will open that reads “Insert plate and press ok”
Insert the plate and then press ok
Save data as before, and e-mail the data to yourself. You may wish to consult
the lab manual for the Riboflavin experiment.
IV.
Analyze the data
Check for outlying data points (this assay can give rather varied
results)
Use Microsoft Excel to construct a standard curve of A595 vs. µg protein.
Remember to do the linear regression analysis.
Use the equation from the linear regression to determine the amount of
protein in wells H1, H2, and H3.
Then calculate the protein
concentration of your unknown BY THAT I MEAN THE
CONCENTRATION IN THE TUBE WITH THE NUMBER, recall
that you diluted it when placing it in the plate. Express the answer in
µg/mL.
Make sure to include the number of the unknown.
Why isn’t water used to blank the
spectrophotometer in this
experiment?
Quantitative Determination of Proteins STUDY GUIDE
1. In this experiment, the “blank” was different from that used in the riboflavin
experiment. How does one know what should be in the “blank”?
2. Suppose you used 0.1 mL of your BSA solution of unknown concentration in the
Bio-Rad assay. If the assay tube had a total volume of 5.1 mL, and the absorbance
indicated a concentration of 10µg protein per tube, what is the concentration of the
unknown BSA solution in µg/mL?
3. Give examples of nitrogen-containing molecules that might be present in a protein
solution.
4. Describe how the Lowry test is a modification of the biuret test. Compare the
sensitivities of these two tests for proteins.
5. There are advantages and disadvantages associated with all of the quantitation
methods discussed in this week’s experiment. When might it be more advantageous
to use the Lowry rather than the Bio-Rad assay? Why?
6. Should there be any concerns associated with the disposal of any of these reagents?
Which one(s)? Why?
7. The molar extinction coefficient of Substance A is 18,550 M-1cm-1 at pH 8.5 and 650
nm. You have 250 mL of Solution A of unknown concentration. When 4 mL of
your Solution A is poured into a cuvette 1 cm wide, 1 cm long, and 4 cm high, and
placed in a spectrophotometer, an absorbance of 0.358 is obtained at pH 8.5 and 650
nm.
a.
What is the molar concentration of your Solution A?
b.
How many moles do you have in 250 mL of solution?
c.
If your 250 mL of Substance A was prepared by dissolving 2.58 x 10-3 grams of
Substance A in 250 mL of solution, what is the molecular weight of Substance
A?