The document describes several experiments related to protein analysis: 1. It provides procedures for preparing phosphate and acetate buffers, estimating protein concentration using Lowry's method, determining the absorption maximum of riboflavin, performing paper chromatography to separate amino acids, and titrating acetic acid to find its pKa value. 2. It also outlines protocols for purifying protein from a sample via ammonium sulfate precipitation and estimating the precipitated protein, separating amino acids from a mixture using ion exchange chromatography, and performing SDS-PAGE to separate proteins by molecular weight. 3. Reagents, materials, and step-by-step procedures are described for each experiment to analyze different properties of proteins and separate mixtures of

2. NAME OF THE EXPERIMENTS
1. BUFFER PREPARATION
2. ESTIMATION OF PROTEIN BY LOWRY’S METHOD
3. ABSORPTION MAXIMA
4. PAPER CHROMATOGRAPHY
5. PH TITRATION
6. PROTEIN PURIFICATION BY AMMONIUM SULFATE
PRECIPITATION
7. ION EXCHANGE CHROMATOGRAPHY
8. SDS – PAGE
9. MOLECULAR MODELLING
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3. BUFFER PREPARATION
AIM:
To prepare phosphate buffer (pH=7.5) and acetate buffer (pH=5).
PRINCIPLE:
A buffer is a solution which reacts which resists small change in hydrogen
concentration of acid or base. Buffer solutions are the mixture of weak acids and their salts
or conjugate base. The conjugate base provided by salt dissociation is actually involved in
the following buffering action.
The capacity of a buffer to resist the change in pH depends on the actual
concentration of a buffer. Salt to acid concentration ration when it’s utility say 1:1 the buffer
has maximum efficiency.
The buffering range of any given buffer is in PH units on either side of the pKa of the buffer
acid.
REAGENTS REQUIRED:
Sodium hydrogen phosphate (NaH2PO4) – acid – 0.1M
Disodium hydrogen phosphate (Na2HPO4) – Base – 0.1M
Sodium acetate (CH3COONa) – acid – 0.1M
Acetic acid (CH3COOH) – Acid – 0.1M
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4. PROCEDURE:
PHOSPHATE BUFFER
• 0.1M of NaH2PO4 and Na2HPO4 solutions were prepared separately.
• To 50mL of 0.1M Na2HPO4, add 0.1M NaH2PO4 and check pH using pH meter.
• The pH was brought to 7.5 by using 0.1M NaH2PO4 solution and the entire volume
was made upto 500mL using distilled water.
ACETATE BUFFER:
• 0.1M CH3COONa and CH3COOH solution is prepared separately.
• To 50 mL of 0.1M CH3COONa 300ml of distilled water is added and its pH checked by
a pH meter.
• The pH is brought to 5 by using 0.1M CH3COOH solution. The final volume is made
500mL with distilled water.
RESULT:
500mL of 0.1M phosphate buffer at pH 7.5 and 0.1M acetate buffer of 500mL volume
at pH 5 were prepared separately.
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5. ESTIMATION OF PROTEIN BY LOWRY’S METHOD
AIM: To estimate the amount of protein in a solution.
PRINCIPLE:
Sodium tungstate, Molybdate and phosphate present in folin ciocateau reagent
reacts with phenolic group of tyrosine residues in protein to produce a blue purple colour.
This has an absorption maximum at 640nm. This method is sensitive upto approximately 10
g/mL of protein concentration. This assay is also sensitive to pH. (Working range pH 10 –
10.5) and length of incubation time.
REAGENTS:
Solution 1: 2% of Sodium carbonate (w/v) keep in 0.1 NaOH
Solution 2: 4% NaK tartarate (w/v) in distilled water
Solution3: 2% CuSo4 in distilled water
Folin ciocateau 50ml in 50mL of water
Bovine serum albumin (BSA) standard: 400µg/mL
PREPARATION OF ALKALINE REAGENT (ALKALINE COPPER)
The solution 1, 2, 3 are mixed in the ratio of 100:1:1 to prepare alkaline reagent.
FOLIN CIOCALTEAU REAGENT:
Commercially available reagent is diluted to 50% by distilled water and used.
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6. BSA STANDARD SOLUTION:
4mg of BSA is dissolved in 1ml of distilled water to get a concentration of 400µg/mL.
PROCEDURE:
• To a series of labelled (Duplicated) testes s1 – s5. BSA solution is added in the range
0.1 mL– 0.5mL.
• The tubes are made up to 0.5mL by adding distilled water.
• The tube with 0.5mL of distilled water serves as blank.
• The 5mL of alkaline copper reagent is added to all the tubes including test which
contains 0.1mL of test sample, 0.4ml of distilled water.
• All the tubes are incubated at room temperature for 20 minutes.
• After the incubation 0.5mL of Folin’s reagent is added to each tube and re-incubated
for 15minutes.
• Absorbance is noted by setting the spectrophotometer to 0% absorbance for blank.
• A standard graph is plotted by tracing the O.D values on Y - axis and increasing by
X - axis.
• The test samples the OD is spotted on the standard and perpendicular is drawn
towards concentration.
• The concentration of protein in this sample can be calculated using the formula.
Amount of protein in test tube= OD of test x conc. of standard x Dilution factor
OD of standard
RESULT:
Amount of protein present in the given solution according to the graph is ………………….
The amount of protein calculated by the formula is ………………….
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7. ABSORPTION MAXIMA
AIM:
To determine the wavelength of maximum absorption for the sample solution riboflavin.
PRINCIPLE:
Every particle in the universe has the property of absorption of light. If differs from
atom to atom of different elements where they can absorb the light rays maximum. Such
that all of its atom are there in excited state. The wavelength at which, a solution of
substance absorbs maximum amount of light is called its absorption maximum.
MATERIALS REQUIRED:
Spectrophotometry
Riboflavin – 5mg/mL
PROCEDURE:
• The riboflavin solution is taken in a test tube 1mL and to it 14 mL of water is added
to make up to volume of 15mL.
• The absorbance of the sample at different wavelength 260 – 650 nm were taken on
UV visible spectrophotometry.
RESULT:
Maximum absorption of riboflavin was founded at ……………..
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8. PAPER CHROMATOGRAPHY
AIM:
To separate a mixture of amino acids by using a paper chromatography.
PRINCIPLE:
Cellulose in the form of paper sheets makes an ideal support medium where water is
absorbed between the cellulose fibres and form the stationary phase. The mobile phase is
secondary butanol, acetic acid and water in the ratio of 4:1:5.
The different amino acid in the mixture are separated based on the difference in
their relative solubility between the stationary phase and the mobile phase. If is the capillary
action which allows the movement of the solvent and sample in the paper.
REQUIREMENTS:
Whatmann no. 1 filter paper
Solvent system (Butanol: Acetic acid: water in 4:1:5 ratio
Ninhydrin (0.25g in acetone (0.2%)
Distilled water
Amino acid sample
Spot lamp
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9. PROCEDURE:
• The machine direction of the paper is observed and cut into many strips in that
direction.
• The sample containing the mixture of amino acids are spotted with capillary tube
(for maximum precision) on a paper at a distance of about 1 inch away from the
edge to be dipped in the solvent system.
• The spot with amino acid sample is concentrated by repeated spotting and drying.
Then the solvent system is kept in chromatographic chamber which is already
saturated with solvent system vapours.
• The chromatographic paper spotted with amino acid samples is dipped in the solvent
system upto 1 cm from the bottom of chamber.
• The solvent is made to run under capillary action such that it travels 2/3 portion of
the paper. The paper is then removed and exposed to NH3 vapours for neutralization
and allowed to dry.
• After drying 0.2% Ninhydrin is sprayed on the dried chromatogram and purple
coloured spots are observed on the chromatogram.
• The distance travelled by the solvent system and the distance travelled by the
respective amino acids from the point of spotting is scaled and Rf is calculated.
Rf value = Distance moved by the solute D
Distance moved by the solvent
RESULT:
The amino acids were separated in different spaces in chromatogram.
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10. PH TITRATION
AIM:
To estimate the change in PH while titrating weak acid against strong base.
PRINCIPLE:
pH signifies potential of hydrogen atoms. It’s the negative log of hydrogen ions
concentration. Acetic acid is a weak acid, it doesn’t undergo protonation easily as ‘ OH’
never gets completely ionised, along with positive ionic effect of adjacent (CH3) group
attached to carbon atom. The H+ of acid gets neutralized with –OH of strong base. The
amount of NaOH needed to bring about neutralization gives PH of that particular acid.
CH3COOH  CH3COO- +H+
NaOH  Na+ +OH-
CH3COOH+NaOHCH3COONa+ H2O
MATERIALS REQUIRED:
pH meter
0.1M NaOH solution
0.1M acetic acid solution
Beaker
Pipette
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11. PROCEDURE:
 Take 10mL of 0.1N acetic acid solution in a clean beaker.
 The pH meter is standardised using standard pH or buffer of known pH.
 The pH of acetic acid is noted.
 Then add 1mL of NaOH to 0.1N acetic acid in beaker with constant stirring or mixing
drop by drop of NaOH to CH3COOH. The pH is noted.
 Like above mentioned add 1mL of NaOH at regular interval and pH noted with
corresponding addition of NaOH upto it reaches alkalinity.
RESULT:
pH titration was performed. The pKa of the acetic acid is …………………
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12. PROTEIN PURIFICATION BY AMMONIUM
SULPHATE PRECIPITATION
AIM:
To purify proteins at different concentration of ammonium sulphate and estimate it by
Lowry’s method.
PRINCIPLE:
Basically the protein precipitation depends upon salting out of ammonium sulphate
on addition of salt of ammonium sulphate on addition of salt to protein the shell of
hydration of proteins is removed. This causes the precipitated amount of protein are
estimated by Lowry’s method.
REQUIREMENTS:
Ammonium sulphate 10g, 20g, 30g weight separately.
Protein standard 10mg, 20mg of protein per mL.
Copper sulphate 10%
Sodium potassium tartrate 2%
Na2CO3 2% of Na2CO3 in 0.1N
NaOH
Folin reagent
STANDARD SOLUTION 1mg of BSA in
distilled water.
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13. Coomessive dye reagent
CBB – G250 100mg
Methanol 50mL
Orthophosphoric acid 100mL
Distilled water make upto 1000mL
PROCEDURE:
Three cleaned beakers containing 10%, 20%, 30% ammonium sulphate are taken.
They are added in succession 100ml BSA solution taken in different contents and constant
stirring is done while adding the salt solution. The solutions are then allowed to stand for
1 hour at room temperature.
An adequate of 1.5ml from each beaker is taken and centrifuged at 1000rpm for 510
minutes. The supernatant is discarded.
The pallet is suspended in 10mL of distilled water. The protein content of pallet is
quantitatively estimated by using barfoed’s method. The values are plotted on a standard
graph.
RESULT:
The amount of protein in the given sample by an ammonium sulphate precipitation
is…………….
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14. ION EXCHANGE CHROMATOGRAPHY
AIM:
To separate amino acids by ion – exchange column chromatography using anion
exchanger.
PRINCIPLE:
Ion exchange chromatography can be used for separation of substances which passes
a net electrical charge. Anion exchanger reversibly bind negatively charged compounds
through electrostatic forces whereas positively charged ones interact with cation exchanger.
Different compounds are held by ion exchangers with varying strengths depending upon
charge. Amino acids are amphoteric substances and have a net charge of zero at isoelectric
pH. At pH below isoelectric point the amino acids are positively charged while ate higher pH
they exist as anions. Hence the pH of system will be high, above isoelectric point where
amino acids exists as anions and can be separated on an anion exchanger.
REQUIREMENTS:
Chromatography column
Amberlite IR-200 polystyrene
Ninhydrin reagent
Glass wool
Amino acid mix 2mg of each amino acid per ml of 0.1N HCL at pH 1.0
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15. PROCEDURE:
PREPARATION OF ION EXCHANGER – AMBERLITE- IR 200
Suspend 10g of amberlite IR 200 into sufficient volume of phosphate buffer for
15minutes to ensure that the resin is saturated with chloride.
EQUILIBRATION OF RESINS:
• Suspend the resin in phosphate buffer of pH 7.4 and allow it to stand for 1hour.
• Mount the column upright and pour suspension with the help of glass rod while
tapping column gently.
• Allow the suspension to settle down. Open the outlet and pass two to three volumes
of phosphate buffer (0.1 M, pH 7.4). This will fully equilibrate the resin to pH 7.4.
When only a thin layer of buffer remains at the top of the resins, stop the flow by
closing column outlet.
SAMPLE LOADING:
Open the outlet and let the buffer at the top to drain the column surface. Close the
stopcock.
DEVELOPMENT OF COLUMN:
All the amino acids would be in anionic form at pH 7.4, so would be bound to anion
exchanger. Gradient elution using increasing pH or 0.5M NaOH solution in buffer solution
used to facilitate sequential elution of the bound amino acids.
RESULT:
The given amino acid mixture were separated by spraying Ninhydrin on the amino
acid spots placed on the whatmann No 1 filter paper. Amino acid concentration can be
determined by observing the intensity of purple colour.
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16. SDS – PAGE
AIM:
To perform SDS – PAGE.
PRINCIPLE:
Sodium dodecyl sulphate (SDS) is an anionic detergent which binds to protein and
confers negative charge uniformly along its entire length.
One molecule of SDS binds to two amino acid residues of proteins. As proteins gain uniform
negative charge, so the rate of migration towards anode is totally determined by the
molecular weight.
MATERIALS REQUIRED:
30% ACRYLAMIDE STOCK SOLUTION acrylamide 30g
Bis acrylamide 0.8g
Distilled water 100mL
RESOLVING GEL BUFFER (pH 8.8) Tris buffer base 22.7g of 1.875M
Distilled water 70mL then adjust pH to 8.8
Make upto 100ml using Distilled water.
STACKING GEL BUFFER (pH 6.8) Tris buffer HCL 7.2g of 0.6M
Distilled water 70mL
Adjust pH 6.8 and make the volume upto100mL
POLYMERIZATION AGENTS:
5% APS Dissolve 5g ammonium per sulphate in 100mL of water
TEMED N,N,N’,N’ tetra methyl ethylene diamine used directly from the refrigerator.
ELECTRODE BUFFER (pH 8.2 – 8.4)
Tris base 12g of 0.005M
Glycine 28.8g of 0.192M
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17. Distilled water 200mL
SAMPLE BUFFER (5x)
Stacking gel buffer 5mL
SDS 0.5g
Mercaptoethanol 0.25mL
Bromophenol blue 1mL (0.5% w/v)
Distilled water 10mL store in frozen aliquots.
10% SDS SOLUTION
SDS 10g
Distilled water 100mL
STANDARD PROTEIN
1g of BSA in 1mL of distilled water.
STAINING SOLUTION
(R250) coomassive brilliant blue dye 0.1g
Methanol 40mL
Acetic acid 10mL
Distilled water 50mL DESTAINING
SOLUTION
Methanol 40mL
Acetic acid 10mL
Distilled water 50mL
PROCEDURE
 ASSEMBLING OF GLASS PLATES
o Thoroughly clean the glass plates and the spacers and assemble them properly. o
Hold the assembly together and damp them in upright position. o Apply 2% agar or
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18. petroleum gel around the edges of the spacers to hold them in place and seal the
space between glass plates.
 PREPARATION OF RESOLVING GEL (30mL)
o 13.3mL of Acrylamide stock is added to 8.0mL of resolving gel buffer (pH 8.8)
and 18.1mL of distilled water. o To this mixture, add
5% APS 0.2mL
10%SDS 0.4mL
TEMED 20µL
 STACKING GEL PREPARATION (4%)
o 1.35mL of acrylamide stock solution is added to 1mL of stacking gel buffer of
pH 6.8 and 7.5mL of distilled water. o Then to this mixture, add
5%APS 50µL
10%SDS 0.1mL
TEMED 10µL
o Remove the water from the top of resolving gel and pour the stacking gel mix
and place the comb in stacking gel and allow to set.
 After the formation of the gel, the comb is removed carefully.
 Sample mixed with sample buffer was loaded in wells.
 The electrode were connected and buffer was poured in tank.
 After connection, the power pack was turned on.
 After the sample reached the lower edge, power pack was turned off.
 Gel was removed from plates and kept overnight in staining solution.  Next day,
the gel was removed from staining solution and kept in destaining solution.
 The protein bands only retains the colour.
RESULT:
SDS PAGE was performed.
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19. MOLECULAR MODELLING
Molecular modeling exercise was carried out using the ball &stick method made
available through the modeling kit (1.tarson 2.polylabs molecular modeling kits) supplied to
the students. Each student constructed the model of a compound whose structure is as
depicted in the record work.
Constructions were carried out using color coded kit components that specifically
identified the nature and position of each element within a compound’s chemical structure.
C-C, C-N, and C-X (where X is any element) bond lengths and bond angles were
measured using centimeter scale and the values were noted. The objective of the exercise
was to learn the possibility of constructing chemical structures in relation to their elemental
orientations and positioning within a given compound using constructional materials that
could then be extrapolated to construct and understand similar or more complex chemical
structures through the use of computer programs /bioinformatics tools.
RESULT:
The compound constructed is ………………
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