Expt. No.

8 Date:

ESTIMATION OF PROTEIN BY BRADFORD METHOD

Aim

To estimate the amount of protein in the given sample

Principle

The protein in solution can be measured quantitatively by different methods. The methods
described by Bradford uses a different concept-the protein‘s capacity to bind to a dye,
quantitatively. The assay is based on the ability of proteins to bind to coomassie brilliant blue
and form a complex whose extinction coefficient is much greater than that of free dye.

Materials Required
 Dissolve 100mg of Coomassie-Brilliant blue G250 in 50 ml of 95% EtOH.
 Add 100 ml of 85% phosphoric acid and make up to 600 ml with d H2O.
 Filter the solution and add 100 ml of glycerol, then make upto 1000ml.
 The solution can be used 24 hrs later.
 Protein solution (Stock standard): Weighed accurately 250 mg of bovine serum albumin
and dissolved in distilled water and made up to 50 ml in a standard flask.
Procedure
 Prepare various concentrations of standard protein solutions from the stock solution (say
0.2, 0.4, 0.6, 0.8 and 1.0 ml) into series of test tubes and make up the volume to 1 ml.
 Pipette out 0.2ml of the sample extract in two other test tubes and make up the volume to
1ml.
 A tube with 1 ml of water serves as blank
 Add 5.0 ml of coomassie brilliant blue to each tube and mix by vortex or inversion.
 Incubate for 10-30minutes and read each of the standards and each of the samples at
595nm.
 Plot the absorbance of the standards verses their concentration. Plot graph of optical
density versus concentration.
Result: The standard graph for protein is plotted, and the unknown conc. is found to be
 Tabulation

Time of Optical
Sample Concentration in ln (1/1-R)
Sonication density (595 R= Pr/Pm
No. mg/ml
(secs) nm)

1.

2.

3.

4.

5.

6.

7.

8.
Expt. No. 9 Date:

CELL DISRUPTION BY SONICATION

Aim
To rupture microbial cells at different time intervals and to measure the amount of protein
released.
Principle
The treatment of microbial cells in suspension with inaudible ultra sound (greater than
18000 Hz) results in their inactivation and disruption. Ultrasonication utilizes the rapid
sinusoidal movement of a probe within the liquid. It is characterize by high frequency (18 kHz
-1 MHz), small displacements (less than 50μm); moderate velocities (a few ms -1), steep
transverse velocity gradient (up to 4000 s1) and very high acceleration (up to 80,000g). In
Ultrasonication phenomena, when acoustic power input is sufficiently high will allow the
multiple productions of micro bubbles, at nucleation sites in the fluid. The bubbles grow during
the rarefying phase of sound wave, and then are collapsed during the compression phase. On
collapse, a violent shock wave passes through the medium. The whole process of gas bubble
nucleation, growth and collapse due to action of intense sound wave is called cavitation. The
collapse of the bubble converts sonic energy to mechanical energy in the form of shock waves
equivalent to several atmospheric (300MPa) pressure. This energy input imparts motions to
parts of cells which disintegrate when their kinetic energy content exceeds the cell wall strength.
An additional factor which increases cell breakage is the micro streaming (very high velocity
gradient causing shear stress) which occur, near radically vibrating bubbles of gas, caused by the
ultrasound.
Much of the energy absorbed by all suspensions is converted to heat effectively, so
cooling is necessary. The rate of protein released by mechanical cell disruption, usually sound to
the proportional to the amount of releasable protein.
dP
  KP
dt
Where P = protein content remaining in associated cells
t = time
K= release constant dependent on the system.
Integrating from P = Pm (maximum possible protein release at time zero) to P = Pt at time ‘t’
gives

Pt t
dP P 
P P   K 0 dt  ln m   Kt
m  Pt 
As protein (Pr) released from the cells is given by Pr =Pm-Pt , the following equation for cell
breakage is obtained.
 Pm 
ln   Kt
 Pm  Pt 
 1 
ln   Kt
1 R 

r m
Where R= P /P

 1 
ln   Kt
1 R 
The constant (K) is independent of cell concentration up to high levels approximately
proportional to the acoustic power above the threshold necessary for cavitations.
Procedure:
1. Take 10ml of culture in 8 eppendrof tubes.
2. The samples are sonicated at different time intervals (Say20sec, 40, 60, 80, 100, 120,140
& 160sec). Place the samples under ice flakes during sonication. Set the power at 50W
and frequency at 60MHz.
3. The samples are spun down at 10,000rpm for 5minutes and 100μl of the supernatant is
transferred to fresh test tubes.
4. Add 900μl ddH2O to the supernatant (1:10 dilution) and 20µl of this is taken for protein
estimation using Bradford’s reagent. The final volume in each well is 200µl (180µl
reagent and 20µl diluted supernatant).
5. Read the OD at 595 nm.
6. Standard was plotted using 5, 10, 15 and 20μl of 0.2mg/ml BSA.
7. A graph of protein content vs. sonication time was plotted.
Result
The highest protein release was obtained when the sample was sonicated for secs.