Sudip Maji 2017ME10616

Subham Kumar 2017CH10251

SBL100 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.

1. How does Bradford dye interact with proteins?

Ans. The Bradford Dye is used to determine the concentration
of proteins in solution. The procedure is based on the formation of a
complex between the dye, Coomassie Brilliant Blue (CBB),
and proteins in solution. At pH 7.4, -vely charged dye interacts with
+vely charged amino acids (of proteins) to form Blue colored complex.

2. Write the principle of spectrophotometer and mention the significance of

using blank?

Ans. A Spectrophotometer works on the principle of Beer- Lambert Law.

It states that the absorbance is directly proportional to the path length of the sample
(the width of the cuvette) & to the concentration of the sample i.e.,

Aαlc

Where, A = Absorbance, l= Path length of sample , c = Concentration of sample.

Significance of Using Blank

Blank is used so as to calibrate the Spectrophotometer to a zero reference. This is to

subtract the individual absorbance of PBS and dye so as to get absorbance
corresponding only to protein-dye complex.
 3. Specify the role of following components.

a. Role of PBS – It helps to maintain a constant pH of 7.4 throughout as well as used

for dilution.
b. Role of BSA – Borine serum albumin (BSA) is used as a standard concentration in
the experiment.
c. Role of PEB – This is used for disruption of cell and to extract total protein from plant
cell.

4. Plot a standard curve for the known protein (BSA), also show the R2 and linear equation
on the graph?

Absorbance vs Conc.
0.9

0.8 R² = 0.9196
0.712
0.684
0.7 0.653
0.596
0.6 0.53

0.5 0.447
Abs.

y = 0.0172x + 0.1162
0.4 0.332

0.3
0.181
0.2

0.1
0
0
0 5 10 15 20 25 30 35 40 45
Conc.( g/ml)

5. Fill the table given below with the results from unknown protein estimation.

At λ=595 nm
SAMPLE SAMPLE VOL. Absorbance of Conc. Of Protein Total Conc.,
Unknown from Standard X= x * D.F.
Protein (Aver.) curve, x ( g/ml) ( g/ml)
Extracted 10 µL 0.636 30.19 6.038
Protein (Fresh)
40 10 µL 0.527 23.88 4.776
RT 10 µL 0.389 22.62 4.523
 𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒
D.F = 𝑆𝑎𝑚𝑝𝑙𝑒 𝑉𝑜𝑙𝑢𝑚𝑒
= 200
6. Specify the ideal order of protein concentration as compared to your observations and
defend your results?
Ans. Ideal order of protein concentration F > RT > 40
where F= Conc. Of Fresh Sample
RT= Conc. At Room Temp.
40= Conc. At 4 0C

This is so because the enzymes (peptidases and proteases) chop down proteins to decrease
their conc. over time. For fresh sample there will be no time for enzyme activity while the
enzyme activity is highest at room temp so leads to the above order.

According to the observations we obtained ideal order of protein concentration.