BCM 6217

Report Sheet No. 2

Group No. 1 Year & Section: 3B - BC

Experiment 2 - Quantitative Analysis

Part 2

I. Pre-lab Questions
1. What is the mechanism of the reaction of the Bradford reagent with proteins?
The Bradford reagent contains an acidified Coomassie Brilliant Blue
G-250 dye that binds to denatured proteins in a solution. A color shift, usually
from brown to blue, in absorbance maximum moving from ~465 to ~595 nm will
be used to determine the concentration of proteins. The absorbance of a dilution
series of protein standards with known concentrations, such as bovine serum
albumin (BSA) and bovine ℽ-globulin (BGG), will be used to determine the exact
amount of proteins in the solution through interpolation from a standard curve.

2. What is the relevance of measuring absorbance against protein concentration?

The simplest and most direct method for determining protein
concentration in solution is to measure absorbance at 280 nm (UV range). Amino
acids with aromatic side chains (such as tyrosine, tryptophan, and phenylalanine)
absorb a lot of UV light. Proteins and peptides absorb UV light in proportion to
the number of aromatic amino acids and total concentration. Since no additional
reagents or incubations are required, directly measuring absorbance is a quick
and convenient way to quantify protein. There is no need to prepare a protein
standard, and the procedure does not consume the protein. The
absorbance-to-protein concentration relationship is linear.

3. What is the difference of measuring proteins through UV-VIS spectroscopy at 280

nm and at 595 nm with the presence of the Bradford reagent?
Tyrosine and tryptophan in particular show a highly selective absorption at
280 nm, enabling direct measurement of A280 protein content. UV absorbance at
280 nm is frequently used in laboratories to determine protein concentration
since it is straightforward, convenient to use, and reasonably priced. To calculate
protein concentration, Basic Protocol 1 compares absorbance at 280 nm (A280)
to a standard curve or known absorptivity values for that protein (a280). The
Alternate Protocol uses absorbance at 205 nm (A205) to compute the protein
concentration since proteins absorb a lot of light at 280 nm due to their three
different types of amino acids. Peptide amino acid bonds also absorb light at 205
nm. The UV absorption of proteins can be used to take non-destructive photos
and collect the spectra of small items. The easiest and most direct test method
for figuring out how much protein is in a solution is measuring the absorbance at
 280 nm (UV range). The UV absorption strategy is not the best one for protein
combinations since different proteins have different amounts of aromatic amino
acids.

II. Data Sheet

Test Tube Volume di Volume 100 Final protein Absorbance Corrected
H2O (mL) mg/mL BSA concentratio at 595 nm Absorbance
(mL) n (mg/mL)

BLANK 1 0 0 0.120 0

1 0.9 0.1 10 mg/mL 0.144 0.024

2 0.8 0.2 20 mg/mL 0.170 0.050

3 0.7 0.3 30 mg/mL 0.192 0.072

4 0.6 0.4 40 mg/mL 0.231 0.111

5 0.5 0.5 50 mg/mL 0.244 0.124

6 0.4 0.6 60 mg/mL 0.265 0.145

7 0.3 0.7 70 mg/mL 0.274 0.154

8 0.2 0.8 80 mg/mL 0.275 0.155

9 0.1 0.9 90 mg/mL 0.285 0.165

Unknown 72 mg/mL 0.268 0.148

protein

Standard curve

y = 0.0018x + 0.0185
R² = 0.9304
 Unknown Protein = 0.268 or 0.148

Concentration of Unknown Protein

y= mx + b
y = 0.0018x + 0.0185
0.148 = 0.0018x + 0.0185
x = 71.94 mg/mL or 72 mg/mL

III. Post-lab Questions

1. What are possible sources of error when preparing a standard curve that has an
R2 value below 0.97?
To create a link between a dependent variable (plotted on the y-axis) and
an independent variable, a standard curve or calibration curve is utilized (plotted
on the x-axis).
Concentrations, measurements, improper data recording, experimental
mistakes, and including the point (0,0) in the graph even though the subsequent
observation has not been taken are some potential sources of error. The line will
fit poorly towards point 0 if such data are arbitrarily inserted, which will influence
the R-squared value.

2. Differentiate the following colorimetric methods based on mechanism, sensitivity

(accuracy of results), and limit of detection (minimum standard concentration):
a. Biuret
The reagent does not contain biuret [(H2N-CO-)2NH]. The test is
so-called because it detects peptide-like bonds in biuret. Copper(II) binds
protein peptide nitrogen atoms in this assay. Copper(II) is reduced in a
secondary reaction (I). Tris and ammonia buffers interfere with this assay,
making it unsuitable for ammonium sulfate-precipitated protein samples.
Due to its insensitivity and low interference from free amino acids, this
assay is best for high-protein whole tissue samples. Detects peptide
bonds.

b. Bradford
The Bradford assay uses Coomassie brilliant blue G-250 to
measure protein absorbance. Coomassie brilliant blue G-250 is cationic,
neutral, and anionic (red). When exposed to acid, the red dye turns blue
and binds to the assayed protein. The solution remains brown in the
absence of protein. Through van der Waals force and electrostatic
interactions, the dye forms a noncovalent complex with the carboxyl and
amino groups of the protein. The red form of Coomassie dye donates its
free electron to the ionizable groups of the protein, disrupting its native
state and exposing its hydrophobic pockets. Through van der Waals
forces, these pockets in the protein's tertiary structure bind non-covalently
to the dye's non-polar region, positioning the positive amine groups near
 the dye's negative charge. The interaction of ions strengthens the bond.
The absorbance shifts from 465 to 595 nm when the dye binds to the
protein. The Bradford protein assay is less prone to interference from
chemicals such as sodium, potassium, or carbohydrates such as sucrose.
Exceptions include detergent concentrations. SDS, a common detergent,
is used to lyse cells and denature proteins for SDS-PAGE by disrupting
the membrane lipid bilayer.

c. Bicinchoninic Acid
The bicinchoninic acid (BCA) assay is a copper-based colorimetric
assay for total protein quantification. Cu2 +-protein complexes form in
basic environments, followed by Cu2 + reduction to Cu+. Protein
concentration reduces Cu2+. Two molecules of BCA chelate to each Cu+
ion, causing a green-to-purple color change and a strong 562 nm
absorbance. A number of peptide bonds and the presence of amino acid
side chains affect the bicinchoninic Cu+ complex. Temperature increases
amino acid exposure and reduces differences in protein amino acid
composition. To increase assay sensitivity, run it at 60 °C. It's
incompatible with most detergents and protein buffers. The detergent in
the B-PER reagent may interfere with your Bradford assay, especially in
your CL sample. The sample was diluted with PBS before affinity
chromatography and again before the assay.

References:

Bio-Rad.com. (n.d.) Bradford protein assay.

https://www.bio-rad.com/featured/en/bradford-protein-assay.html#:~:text=The%20Bradfo
rd%20protein%20assay%20is,from%20470%20to%20595%20nm.

Measuring protein concentration using absorbance at 280 nm. (n.d.). Rice University -- Web
Services. https://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html

Miller A, Jedrzejczak WW. Albumina--funkcje biologiczne i znaczenie kliniczne

[Albumin--biological functions and clinical significance]. Postepy Hig Med Dosw.
2001;55(1):17-36. Polish. PMID: 11355530.

Murphy, P. A. (2008). Soybean Proteins

https://www.sciencedirect.com/science/article/pii/B97818939976465001

Overview of protein assays methods. (n.d.). Thermo Fisher Scientific - US.

https://www.thermofisher.com/ph/en/home/life-science/protein-biology/protein-biology-lea
rning-center/protein-biology-resource-library/pierce-protein-methods/overview-protein-as
says.html
The Protein Man. (n.d.). Spectrophotometry and its application in protein estimation.
https://info.gbiosciences.com/blog/bid/201326/spectrophotometry-and-its-application-in-p
rotein-estimation

What is gluten—Why is it special? (n.d.). Frontiers.

https://www.frontiersin.org/articles/10.3389/fnut.2019.00101/full