SBL100 LABORATORY

Name & Entry No.: Wednesday 1-3 slot

1) Jay Prakash Patidar (2015CE10331)
2) Kuldeep Kumar Meena (2015ce10335)

AIM: To understand how to visualize and/or measure biomolecules

in this case proteins.
INTRODUCTION:

General introduction: Proteins are large biomolecules, or

macromolecules, consisting of one or more long chains of amino acid
residues. CBB Dye (Coomassie Brilliant Blue ) used to visualize proteins
via a regressive staining approach in which gels are saturated with dye
and then destained with an aqueous solution.

Principal: The Bradford assay, a colorimetric protein assay, is based on

an absorbance shift of the dye Coomassie Brilliant Blue G-250. Under
acidic conditions the red form of the dye is converted into its bluer
form, binding to the protein being assayed. The dye forms a strong,
noncovalent complex with the protein's carboxyl group by Van der
Waals force and amino group through electrostatic interactions.
 During the formation of this complex, the red form of Coomassie dye
first donates its free electron to the ionizable groups on the protein,
which causes a disruption of the protein's native state, consequently
exposing its hydrophobic pockets. These pockets in the
protein's tertiary structure bind non-covalently to the non-polar region
of the dye via the first bond interaction (van der Waals forces) which
position the positive amine groups in proximity with the negative
charge of the dye. The bond is further strengthened by the second
bond interaction between the two, the ionic interaction. The binding of
the protein stabilizes the blue form of the Coomassie dye; thus the
amount of the complex present in solution is a measure for the protein
concentration, and can be estimated by use of an absorbance reading.
*Beer Lambert law: BeerLambertBouguer law relates
the attenuation of light to the properties of the material through which
the light is travelling. It is linear relation between absorbance and
concentration of an absorbing species.

MATERIAL AND REAGENTS USED:

Material Required:
1) Mortar pestle
2) Beaker
3) Micro pipet
Reagent required:
1) BSA(Bovine serum albumin)
2) CBB Dye(Coomassie Brilliant Blue G-250)
3) Buffer(PBS-Phosphate-buffered saline, PEB-Protein Extraction
Buffer)
4) Ethanol
 5) Water
METHEDOLOGY:
1) First find the standard plot for absorbance and concentration.
2) Find the absorbance of unknown solution.
3) Using the standard plot find concentration of protein.

OBSERVATION AND RESULTS:

Observation-
Conc of BSA Vol of BSA (ul) Vol of PBS(ul) Abs.
(ug/ml)

0 0 400 0
5 10 390 0.192
10 20 380 0.338
15 30 370 0.406
20 40 360 0.54
25 50 350 0.633
30 60 340 0.653
35 70 330 0.703
40 80 320 0.735

Abs Reading of Leaf-

Fresh = 0.244
At Room temperature = 0.189
At 40C = 0.179
 Standard plot-
Abs V/s Conc
1
0.9 y = 0.0217x
0.8 R = 0.8656
0.7
0.6
Abs.

0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25 30 35 40 45
Concntration of BSA(ug/ml)

CALCULATION-
Y=0.0217x
Abs= 0.0217 Conc.
Dilution factor (DF)=(Final Volume)/ (Initial Volume)=(2000)/10 = 200
Conc. Of Protein = X * 200
Protein Abs Concentration(ug/ml)
Fresh condition 0.244 2248.82
At RT 0.189 1741.94
At 40C 0.179 1649.77

Order of Protein Conc.

Fresh> RT > 40C
Generally order should be Fresh > 40C >RT
But our order differ because we kept the protein extract for 2 weeks and it get
contaminated so protein conc. At room temp. increased w.r.t. 40C.