Name & Entry No: Sumanth Gaduputi 2017EE10451

Rohan 2015EE10475

SBL100 LABORATORY
Laboratory on Protein Estimation
Aim: To understand how to visualize and “measure”
biomolecules – in this case, “proteins”.
Methodology: (1) Operations of a spectrophotometer,
calibration of the spectrophotometer for “Bradford Assay”.
(2) Extraction of “crude extracts” from
biological samples for estimation of total protein content.
Answer the following questions
1. How does Bradford dye interact with proteins?
Bradford dye is scientifically called Commassie Brilliant
Blue. The principle of this is that the binding of protein
molecules to Coomassie dye under acidic conditions results in
a color change from brown to blue. The dye forms a strong
non-covalent complex with the protein’s carboxyl group by
VanderWaal’s forces and amino group through electrostatic
interactions.
2. Write the principle of spectrophotometer and mention the
significance of using blank?
The spectrophotometer is an instrument which measures
an amount of light that a sample absorbs. The
spectrophotometer works by passing a light beam through
a sample to measure the light intensity of a sample.
A = εlc
where A is absorbance (no units)
ε is the molar absorbtivity with units of L mol-1 cm-1
(formerly called the extinction coefficient)
l is the path length of the sample, usually expressed in cm
c is the concentration of the compound in solution,
expressed in mol L-1

A BLANK is used to calibrate the Spectrophotometer so

that any absorbance attributable to the solvent and/or
glass cuvette can be compensated. By zeroing the
Spectrophotometer to the blank, an instrumental
subtraction will measure only the absorbance due to the
substance which is protein in this case.
3. Specify the role of following components
a. PBS: Phosphate Buffer Saline is used to maintain the
pH of the solution thereby maintaining protein stability.

b. BSA: Bovine Serum Albumin is a protein extracted

from cattle. It is used to make a standard curve for
absorbance vs concentration and later this curve is used to
estimate the concentration of unknown protein.

c. PEB: Protein Extraction Buffer is used to extract the

protein of Ashoka leaf in the second part.
4. Plot a standard curve for the known protein (BSA), also
show the R2 and linear equation on the graph?

Absorbance vs. Concentration

0.9
0.8 y = 0.0178x + 0.112
R² = 0.9065
0.7
0.6
Absorbance

0.5
Absorbance
0.4
Trendline for Absorbance
0.3
Linear (Absorbance)
0.2
0.1
0
0 10 20 30 40 50
Concentration(microgram/ml)

5. Fill the table given below with the results from unknown
protein estimation and show calculations and mention
their units?

Sample Volume Absorbance Concen Total Amount

of of protein tration Concen of
protein( (microgram Of tration protein
microlitr /ml) protein (C*DF) in
e) (from (mg/ml) sample(
curve) mg)
Extract 10 0.505 21.88 4.376 4.376
ed
protein
Room 10 0.466 19.88 3.977 1.988
temper
ature
4° C 10 0.557 25 5 2.5

*DF- Dilution factor

6. Specify the ideal order of protein concentration as
compared to your observations and defend your results?
EXPECTED ORDER: FRESH SAMPLE > SAMPLE AT 4° C >
SAMPLE AT ROOM TEMPERATURE.
But the trend observed is:
SAMPLE AT 4° C> FRESH SAMPLE > SAMPLE
AT ROOM TEMPERATURE
The deviation from the expected trend may be due to
pipetting error or action of microbes on protein . Therefore
the following trend is observed.