Experiment-1

BIO F110 BIOLAB 2023-2024

Protein Lab
Measurement of total protein content in the given
sample by Lowry’s method
 Proteins
• Make up about 15% of the cell and have major role on the growth
and maintenance of the human body
• Have many functions in the cell:
⮚ Enzymes
⮚ Transport
⮚ Storage
⮚ Signaling
⮚ Gene regulation etc.

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 Protein Structure
• Proteins are highly complex macromolecules and made up of amino acid residues (20 amino acids) .
• Individual amino acid residues are joined by peptide bonds to form the linear polypeptide chain.
• This linear polypeptide chain is folded into specific structural conformation or simply “structure”.

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 Principle of Spectrophotometric method
(Figure to be explained with instrument)

What is Beer’s law:

Beer’s law was stated by August Beer which states that concentration and absorbance are directly proportional to each other.

What is Lambert’s law?

Johann Heinrich Lambert stated Lambert law. It states that absorbance and path length are directly proportional.

Beer-Lambert’s law:
The combined form of both these law is termed as Beer-Lambert’s law, which states that, for a given material sample path
length and concentration of the sample are directly proportional to the absorbance of the light.
where,
•A is the amount of light absorbed for a particular wavelength by the sample
•ε is the molar extinction coefficient
•L is the distance covered by the light through the solution
•c is the concentration of the absorbing species BIO F110
 Colorimetric estimation of total protein content:
Lowry’s method:

• Age old method for protein estimation.

• Principle of this method is based on two reactions leading to color complex formation.
• First reaction: Biuret reaction, in which Cu2+ ions of the reaction mixture reacts with peptide bond of
proteins under alkaline condition resulting in their reduction to cuprous ion Cu+.
• Second reaction: Lowry’s reaction in which Folin-Ciocalteu reagents, which contain phosphomolybdic
complex which is a mixture of sodium tungstate, sodium molybdate and phosphate along with copper
sulphate solution and protein forms a blue purple color which can be assessed by measuring the
absorbance at 700-740nm

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 Lowry’s method of protein estimation

o Advantage:
• Highly sensitive and gives accurate result
• Require less amount of protein
o Disadvantage:
• Many common substances, such as K+, Mg2 +, NH4+, EDTA, Tris-HCl, carbohydrates, and
reducing agent interfere with this method.

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 Reagents Required:
1. Reagent A: Na2CO3 +NaOH
2. Reagent B: CuSO4. 5H2O + Sodium Potassium tartrate (C4H4O6KNa · 4H2O)
3. Reagent C: 49 ml of reagent A + 1 ml of Reagent B
4. 500 ml reagent C (for 10 groups) to be freshly prepared by Naresh and 50 ml to be given to
each group by instructor
5. Reagent D: 1N Follin-Ciocalteu reagent (Will be available on working bench)
6. BSA stock solution of (1 mg/ml) as a protein standard
7. 5ml BSA working solution prepared by diluting stock 5 times (200 microgm/ml) will be
provided by the instructor for every batch
8. Test sample is to be given from working BSA stock
9. Graph paper will be provided
 Work flow

Test BSA Water(ml) BSA Final Reagent Incubate Reagent D Incubate Ab

Tube (ml) Conc.(ug/ml) C(ml) @RT or Follin @ RT in @740nm
reagent dark

B -- 1.0 0.00 5 10min 0.5 20min

S1 .25 .75 50.00 5 10min 0.5 20min
S2 .50 .50 100.0 5 10min 0.5 20min
S3 .75 .25 150.0 5 10min 0.5 20min
S4 1.0 -- 200.0 5 10min 0.5 20min
T -- 1.0 ? 5 10min 0.5 20min
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 Observation

Test Tube No Conc. of Protein OD @740nm

B
S1
S2
S3
S4
T

Result:
• Plot the values of ID 740 nm against diff conc. Of std protein solution in the graph paper for
preparation of std curve
• Match the OD of T on the std curve. The corresponding conc. on the x-axis will be the actual
protein conc.(ug/ml) in the given sample.

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8 Total glass
test tubes
and a stand
 Operational planning
• Ask students to submit 2 test tubes/group (1 for protein std + 1 for unknown
sample)
(1) 5 ml protein standard (200µg/ml) to be given to each group
(2) 1 ml of unknown standard to be given per group
(3) 50 ml of reagent C per group in reagent bottle (Will be prepared by Naresh)
(4) Water will be available in wash bottle with each working bench
(5) Reagent D will be available at each bench
• Table given in manual to be followed for performing
• To ensure equal participation each students in a group may be asked to
dispense 1 of the 4 requirements in all tubes (Water, protein, Reagent C &
Reagent D and the 5th student can be involved in taking readings ). This way all
students will be working.
Marking Scheme
Attendance: 1 mark (Late -1)
Lab Attire: 1 mark (Improper attire -1)
Performance marks : 5 Marks (Participation in class and lab)
(Not cleaning glassware -1; Breaking Glassware -1)
Results: 5 Marks
● 2 Marks for proper plotting of graph (Neatness, Labeling, etc.)
● Result: - 2 Marks (Give full 2 marks if readings are in ascending order
with almost accurate results (15% error) and 1.5 if final result is not
accurate but readings are in ascending order.
● 1 Mark for 1-2 line of personal interpretation.