GOOD LABORATORY PRACTICES

1. Always follow the experimental protocol or supervisor’s instructions precisely.

2. Wear lab coats inside the lab and remove them before exiting to prevent contamination.
3. Tie back long hair and avoid wearing loose clothing or jewelry during lab work.
4. Use disposable gloves when handling proteins, chemicals, or biological samples; discard
them after use.
5. Maintain a clean and organized workspace to avoid cross-contamination.
6. Do not eat or drink inside the proteomics lab.
7. Avoid touching your face, especially your mouth, eyes, and nose, during lab work.
8. Use only mechanical pipettes; never pipette by mouth.
9. Do not walk around with open tubes, pipettes, or loops containing protein samples.
10. Do not pour chemicals, buffer waste, or solvents down the sink; use proper disposal
containers.
11. Treat all samples and reagents as potentially biohazardous unless proven otherwise.
12. Use trays to transport proteomics reagents, tubes, and buffers safely within the lab.
13. Never handle broken glass directly—use tools like tongs, dustpans, or gloves.
14. Clearly label all vials, tubes, and reagents with sample names, dates, and concentrations.
15. Turn off heating blocks, centrifuges, and water baths when not in use.
16. Keep sensitive samples on ice when required.
17. Segregate and dispose of all lab waste—including gels, solvents, and sharps—using
correct waste bins.
18. Use clean, protein-free cuvettes or microplates for accurate readings.
19. Handle acrylamide with care — it is a neurotoxin in liquid form.
20. Store dye reagent away from light and heat to prevent degradation.

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 INTRODUCTION

Proteomics is the large-scale exploration of the proteome, the dynamic and diverse
collection of proteins expressed by a cell, tissue, or organism at any given time.

Protein estimation is a fundamental step in biochemical and molecular biology research,

as it determines the concentration of proteins in a given sample. Accurate protein
quantification is essential before performing downstream applications like electrophoresis
(SDS–PAGE), enzyme assays, or mass spectrometry. Various colorimetric and
spectrophotometric methods are commonly used to estimate protein content, each based
on specific chemical reactions between the protein molecules and a reagent that produces
a measurable colour change.

Proteomics plays a pivotal role in modern biology and medicine by offering a real‑time
snapshot of cellular function through direct measurement of proteins, the workhorses of
life. Unlike genomics or transcriptomics, which infer function indirectly, proteomics
reveals protein identity, quantity, structure, and interactions, making it indispensable for
discovering early disease biomarkers (e.g., cancer, cardiovascular conditions), drug
targets, and personalized treatment strategies.

Electrophoresis refers to a suite of laboratory techniques used to separate and analyze

charged biomolecules, such as DNA, RNA, and proteins, by applying an electric field to
a medium like gel or capillaries. The core principle hinges on electrophoretic mobility,
which depends on a molecule’s charge-to-mass ratio: when the electric field is applied,
negatively charged molecules (e.g., DNA) migrate toward the anode at rates inversely
proportional to their size, smaller fragments traverse the porous matrix of agarose or
polyacrylamide more quickly than larger ones.
The choice of medium, agarose for bulkier nucleic acids and polyacrylamide for smaller
proteins or oligonucleotides, tailors the resolution and effectiveness of the separation .

Electrophoresis encompasses various techniques tailored to specific analytical needs.

Traditional methods like agarose and polyacrylamide gel electrophoresis separate DNA
and proteins, with SDS‑PAGE focusing on protein size. Advanced formats include
pulsed‑field gel electrophoresis for large DNA, isoelectric focusing for charge-based
separation, and capillary electrophoresis for automated, high-throughput analysis. These
techniques are essential in molecular biology for biomolecule sizing, purification, and
identification in fields like genetics, proteomics, diagnostics, and forensics.

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 PIPETTE HANDLING

Proper pipette handling is fundamental for obtaining accurate and reproducible

experimental results. The user should always select a pipette whose volume range closely
matches the intended transfer volume, ideally, operating between 35–100 % of its
nominal capacity, to maximize precision.

Use new sterile tips for each transfer, aspirate gently to the first stop, immerse the tip just
below the liquid surface, release smoothly, wait a moment, then dispense fully, including
the second stop, while touching the tip to the vessel wall to ensure complete delivery.

Regular calibration, proper cleaning and greasing, and training in consistent timing and
posture are also essential to minimize systematic and random pipetting errors.

Ensure that the pipette tips are compatible for that pipette; Pre-wet the pipette tip; Follow
proper aspiration and dispensing technique; Maintain consistent pipetting speed and
technique for accurate and repeatable results; Maintain and calibrate the pipettes.

Hold the pipette in a vertical position during aspiration, tilt for dispensing.

When multiple aliquots are dispensed, it is recommended to discard the first and final
aliquot.

Choose specialty tips, like extended length or wide orifice ones for the applications that
benefit from them.

Viscous samples should be aspirated and dispensed at slower speeds. In addition, pausing
after every aspiration or dispense gives the liquid more time to smoothly move into or out
of the tip.

Standard (forward) mode pipetting is used for aqueous solutions, which are the majority
of solutions used in laboratory experiments. Reverse mode pipetting is recommended for
liquids with high viscosity or volatility.

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 PROTEIN EXTRACTION
(A) Test 1- Whole earthworm:

1. Harvested healthy earthworms, rinsed thoroughly in water to remove debris

and then blotted dry on filter paper.
2. Weighed approx. 250 mg of washed worms.
3. 1 mL of ice-cold 1X PBS was added.
4. Homogenized on ice with a mortar & pestle.
5. Centrifuged at 10,000–15,000×g for 10–20 min at 4 °C.
6. Collected the supernatant (soluble protein fraction) carefully.

(B) Test 2- Earthworm mucus:

1. Ratio of 1.3 g (earthworm biomass):1 ml (pure water) was added to the beaker
and earthworms were stirred with a glass rod for 15 min (every third minute for 1
min).
2. The extract was centrifuged twofold at 20,000 g at 20 °C for 30 min to remove
remaining mineral particles.
3. Collected the supernatant carefully.

(C) Test 3- Neem leaf:

1. 0.1g of neem leaf was weighed and rinsed thoroughly in water, air dried.
2. 1 mL of ice-cold 1X PBS was added.
3. Homogenized on ice with a mortar & pestle.
4. The sample was vortexed for 1 minute.
5. Centrifuged at 10,000–15,000×g for 10–20 min at 4 °C.
6. Collected the supernatant carefully.

(D) Test 4- Microbes:

1. 1ml of sample was taken in eppendorf.

2. Centrifuged at 10,000×g for 5 mins and discarded the supernatant.

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 3. 1ml of sample was added to the pellet.
4. Centrifuged at 10,000×g for 5 mins.
5. Supernatant was collected and 1ml of 1X PBS was added.
6. Vortexed for 1 minute.
7. Centrifuged at 10,000×g for 5 mins.
8. Collected the supernatant carefully.

Estimation of Protein by Bradford Method

Aim:

To estimate the concentration of protein using Bradford Method.

Principle:

The Bradford protein assay is a commonly used method for estimating the concentration of
proteins in a sample. Generally, it is based on the binding of Coomassie Brilliant Blue (CBB)
dye to proteins, resulting in a shift its maximum absorbance maximum from 465 nm to 595 nm.
The orginal maxium absorbance of CBB is 465 nm and if it binds with the protein the absorbanc
will be shifted to the 595nm resulting in a change in color from brown to blue. The Solution of
CBB is originally brown and after linked with the protein molecule the color will be changed to
blue color complex. The strong noncomplex bond is formed between carboxyl groups of the
protein and dye by van der Waals force and amino group through electrostatic interactions.

The amount of protein In a sample is determined spectrophotometrically by measuring the

absorbance of the blue-colored solution at 595 nm and comparing it to a standard curve
generated using known concentrations of protein.
Materials Required:
Equipments:

• Spectrophotometer
• Glass or polystyrene cuvettes

Chemicals/reagents:

• Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95%

ethanol/Methanol, add 100 ml 85% (w/v) phosphoric acid. Make up to 1 liter after
the dye has completely solubilized, and filter through filter paper before use.
• Protein Standard: 1 mg Bovine serum albumin (BSA) /ml.

Glass wares and others:

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 • Test-tubes
• Pipettes
• Micro centrifuge tubes
• Filter paper
• Funnel

Procedure:

(i) Prepared the aliquots of Pipette out 10ul to 90ul of working standard BSA into the
series of labeled test tubes. And, made up the volume to 100ul in all the test tubes
using distilled water.
(ii) 5ul of the given test samples in another test tubes and labelled as “Test” and 100ul of
distilled water in another fresh tube and labeled as “blank”.
(iii) 2.5ml of freshly prepared Bradford reagent was added to all the test tubes including
the test tubes labeled ‘blank’ and ‘test’.
(iv) Mixed the contents of the tubes uniformly by vertexing / shaking the tubes and
incubated at room temperature for 10- 15 mins.
(v) Finally, the absorbance of all the test tubes was recorded at 595 nm against blank in
the spectrophotometer.

Observation Table:

Estimation of protein by Bradford method

S. No Volume of Volume of Volume of Absorbance

Standard Distilled Bradford (A595nm)
BSA(ul) water(ul) reagent(ml)
Blank - 100 2.5 0

S1 10 90 2.5 0.309

S2 20 80 2.5 0.341

S3 30 70 2.5 0.450

S4 40 60 2.5 0.478

S5 50 50 2.5 0.570

S6 60 40 2.5 0.603

S7 70 30 2.5 0.700

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 S8 80 20 2.5 0.834

S9 90 10 2.5 0.812

SODIUM DODECYL SULPHATE POLYACRYLAMIDE

GEL ELECTROPHORESIS (SDS-PAGE)

AIM:

SDS-PAGE was performed to separate and observe the protein pattern of the sample.

PRINCIPLE:

The principle of SDS‑PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis) is

to separate proteins based purely on their molecular weight by first denaturing them and coating
them with the anionic detergent SDS. SDS binds uniformly along the polypeptide backbone,
masking intrinsic charge and unfolding tertiary structures, while reducing agents break disulfide
bonds, resulting in linear proteins with a consistent negative charge proportional to their length.
These negatively charged chains are then subjected to an electric field and migrate through a
porous polyacrylamide gel; smaller proteins navigate the gel matrix more easily and thus travel
faster than larger ones, enabling precise size-based separation.

REAGENTS REQUIRED:

1. Preparation of stock solution and buffers:

30% acrylamide
a) Acrylamide: 29.2g
b) N, N-methelyne–bis–acrylamide: 0.8g Added water, dissolved and
made upto 100mL and filtered with Whatman no.1 filter paper.
2. Separating gel buffer:
a) Tris-HCl: 1.5M, pH 8.8
18.171g of Tris was dissolved in 60mL of water and adjusted the
pH to 8.8 with HCl and finally made upto 100mL with water.
3. Stacking gel buffer:
a) Tris-HCl: 1M, pH 6.8
6.057g of Tris was dissolved in 60mL water and adjusted the pH to
6.8 with HCl and upto 100mL with water.

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 4. 10% SDS solution: 1g of SDS in 10mL of distilled water.
5. N,N,N’N’-Tetra methylene diammine(TEMED)
6. 10% Ammonium per sulphate (APS): 1g of APS in 10mL of distilled
water.
7. Electrophoresis Buffer:
A) Tris: 25mM, pH 8.3
B) glycine: 250mM,pH 8.3
C) SDS: 0.1%: Dissolved in minimum amount of water (500mL) and
then added SDS.
Allowed to settle and dissolved. This was finally made upto 2.5liters.
8. Staining solution (100mL):

A) Alcohol: 40%
B) Acetic acid: 10%
C) Commassie Brilliant Blue (CBB): 259mg
D) Distilled water: 50%
9. Destaining solution (100mL)
A) Alcohol: 50%
B) Acetic acid: 10%
C) Distilled water: 40%

PROCEDURE:

Preparation of gel:

The glass plates were washed in warm detergent solution, rinsed subsequently in tap water,
deionised water and ethanol and dried. The unnotched outer plates were laid on the table and
Vaseline (or grease) was coated. Spacer strips were arranged approximately at the sides and
bottom of the plates. The notched inner plates were laid in position, resting on the spacer strips
and the arrangement was mounted vertically. Sealing was done properly to avoid leakage.
The volume of the gel solution required for making separating gel was calculated as follows.

Reagents 10%
D.H2O (ml) 2.6
30% acrylamide (ml) 2.1
1.5M Tris (pH 8.8) (ml) 1.6
10% APS (ul) 80
TEMED (ul) 20
APS and TEMED were added just prior to the pouring of gel. The solution was mixed well and
poured into the space between the two plates leaving an inch of the upper space unfilled. Water

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was carefully laid over the surface of the poured gel mixture to avoid air contact, which reduces
the polymerization reaction. The gel mixture was allowed to polymerize, undisturbed at room
temperature for 60 minutes. In the mean time gel mixture for stacking gel was prepared(The
reagents in the following table yield 2.5mL of solution after the addition of APS & TEMED).

Reagents Volume (ml)

D.H2O (ul) 835
30% acrylamide (ul) 625
1M Tris-Cl, pH 6.8 (ul) 975
10% APS (ul) 65
TEMED (ul) 5
After the separating gel was polymerized the over laid water was removed carefully with filter
paper and an appropriate comb was inserted between the plates. 10% APS and TEMED were
added to the stacking gel mixture. It was mixed well and poured immediately (to the brim) over
the separating gel. The stacking gel was allowed to polymerize. Additional gel mixture was
added when gel retracted significantly.

Preparation of protein samples:

An equal volume of loading dye was added to protein samples.

The samples were incubated for 2min in a boiling water bath prior to loading.

When the polymerization was completed the comb was removed and the lower spacer strip was
carefully removed.

The Vaseline (or grease) from the bottom was removed with a piece of tissue paper. The gel was
attached to the electrophoresis tank using appropriate clips/clamps.

The lower reservoir was filled with 1x electrophoresis buffer, using a bent Pasteur pipette or
syringe needle to remove any air bubble trapped beneath the bottom of the gel.

The protein samples along with ladder were loaded using a micropipette and the wells were
completely and carefully filled with 1x electrophoresis buffer.

The upper reservoir was also carefully filled with 1x electrophoresis buffer.

The electrodes were connected to a power pack.

The gel was run at constant current (20 milli ampere 100 volts) for 4-6 hrs at room temperature.
Electrophoretic mobility of the samples was determined by bromophenol blue front. At the end
of the run the power pack was switched off. The gel and plates were laid flat on the table and a
corner of the upper glass plate was lifted up and the gel was carefully removed.

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Staining of the gel:

After the completion of the electrophoresis, the gel was fixed with 10% trichloroacetic acid for
5minutes and stained with Coomassie Brilliant Blue(CBB) . The CBB staining solution was
prepared using methanol, acetic acid and double distilled water in the ratio of 4:1:5 and 0.25gm
of CBB was added and the gel was stained over night.

Destaining of the gel:

The destaining of CBB stained gel was done by using methanol, acetic acid and double distilled
water in the ratio of 5:1:4 till the appearance of clear bands on the gel.
RESULT:
The sample proteins are separated by Sodium Dodecyl Sulphate-PolyAcrylamide Gel
Electrophoresis. The proteins appeared as discrete bands in the gel.

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 FAST (RAPID) COOMASSIE BRILLIANT BLUE STAINING

AIM:

To visualize protein bands in SDS-PAGE gels quickly.

PRINCIPLE:

Coomassie Brilliant Blue R‑250 dye binds noncovalently to proteins in an acidic methanol,
acetic acid (or ethanol–acetic acid) environment through ionic interactions with basic residues
(like lysine, arginine, histidine) and hydrophobic interactions with exposed nonpolar amino acid
regions; the acidic alcohol both denatures and fixes proteins in the gel, enhancing dye binding
and preventing diffusion, while heat or agitation speeds the process, resulting in visible blue
protein bands against a clear background in just minutes instead of hours.

SILVER STAINING

AIM:
To visualize proteins with ultra-high sensitivity, detecting bands in the low‑nanogram range, after
electrophoretic separation on polyacrylamide gels.
PRINCIPLE:

Silver staining for SDS‑PAGE is based on the highly specific deposition of metallic silver at
protein locations: after proteins are fixed and sensitized in the polyacrylamide matrix to remove
interfering substances (like SDS and salts), silver ions selectively bind to certain functional
groups on the proteins (such as carboxyls, histidines, cysteines, and lysines). During
development, these bound Ag⁺ ions are chemically reduced, typically by formaldehyde or other
reducing agents, into visible metallic silver grains clustered at protein bands. The result is

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sharply contrasted, dark-brown to black protein bands with sensitivity in the low nanogram range
due to selective silver reduction proximal to proteins.

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