BASIC METHODS TO

STUDY CELL
BIOCHEMISTRY
Centrifugation
• Itis a process in which a centrifuge machine is used to
separate the components of a complex mixture.
• Principle
•By spinning the sample at high speed, the complex
components of a given mixture are subjected to
centrifugal force which causes more dense particles
to migrate away from the axis of rotation and
lighter ones towards it.
 Centrifugal Force

•It is a force which acts outwards on a

rotating object.
•Greater the speed of rotation, greater will
be the force.
•It is measuredas “g” .
•These particles can sediment at the bottom of the
tube which is called pallet and the remaining
solution, supernatant can be further processed or
analyzed.

•The speed of centrifugation is measured in

rotations per minute, or revolutions per minute
(RPM).
 Applictions of Centifugation
• This method/technique is used to separate or concentrate materials
suspended in a liquid medium.

• Also used in research labs for separation puposes e.g. DNA extraction.

• It is used to separate two miscible substances.

• Fractionation of subcellular organelles can also be carried out.

• Preparation of plasma and blood cells from whole blood.

Applications

• To purify proteins for research or medical use.

• To prepare samples for microscopy.

 Ultracentrifugation
This technique was developed by Svedberg. It is a special technique to spin
sample at exceptionally high speed.
• Principle

• The greater the centrigugal force, the faster the particles sediment.

Large particles can be sedimented at high centrifugal forces while smaller

molecules cannot. For sedimenting smaller molecules of comparable densities,
very high speed and more time is required.

Samples are rotated with the rotational speed of up to 150, 000 RPM.(100,000g).
• This process is carried out in a vacuum to prevent air friction and to

avoid damage of sample at very high speed which causes overheating.

so vaccume also maintains temperature in this case.

• Sedimentation constant is expressed in svedberg units S as rate of

sedimentation depends on size, shape and density of solute particles.

 Applications
• It is a commonly used technique in molecular biology , biochemistry and

cell biology.

• It is used for the separation of small particles like viruses, viral particles,

proteins, protein complexes, lipoproteins, RNA and plasmid DNA.

 pH Metery
It is an electrometric method for determination of pH.

Principle

A typical pH meter consists of a special glass electrode connected to an electronic

meter that measures and displays reading of pH of unknown sample solution. It

detects number of freely present hydrogen ions (protons).

 Priniciple

A typical pH meter is based on exchange of ions from sample solution

to inner solution (7 pH buffer, KCl solution) of glass electrode through

glass membrane that generates electric voltage.

 Applications
• It
is used in blood chemistry investigation (arterial blood
gases) to evaluate acid base disturbance in body.
• It
is used to prepare reagents, buffers of fix value of pH (in
research labs).
• Diagnostic labs rely on pH meters for measurements of
biological samples/fluids pH (blood, CSF, urine) etc.
• Itis also used in pharmaceutical industry during drug
preparation.
• It is also utilized to evaluate environmental parameters and to
monitor water quality.
 Isoelectric focussing
• Inthis technique proteins are allowed to distribute themselves
according to their isoelectric pH values. So, proteins/amino
acids are separated on basis of pI.

•A mixture of low molecular weight organic acids and bases is

allowed to distribute across the gel by subjecting them to an
electric field.

• In this way a pH gradient is established.

When mixture of protein is applied to the gel, the
proteins start migrating but their further migration
stops when they reach the region of gel having pH
equal to their isoelectric pH values so different
proteins can be identified.
Application:
More better resolution of proteins is obtained by
combining this technique with SDS PAGE that is
known as two dimensional gel electrophoresis.
CHROMATOGRAPHIC SEPERATION TECHNIQUES

Chromatographic separations are classified by the chemical or physical means

used to separate the solutes.

•Ion exchange.

•Partition.

•Affinity.

•Adsorption.

•Gel filtration
 Principle
• It
is a technique used to separate group of similar sbstances on the
basis of differences in certain physical characteristics.
• Inevery chromatographic separation, there is a stationary phase
which remains fixed in a system or it consists of packing within a
column.
• The other phase is mobile phase which percolates over the surface of
the fixed phase.
• Stationary phase is usually water or any polar solvent and mobile
phase is relatively non-polar. Stationary phase is liquid supported on
silica gel, cellulose, starch, in sheet form, when cellulose is in form of
paper, it called as paper chromatography.
Rf value

•Rf value=
• Distance travelled by solute
(cm)/ Distance travelled by
solvent (cm) × 100
 Paper Chromatography
Stationary phase is a layer of water or polar solvent coated on the paper fibers.
Mobile phase is a mixture of organic and inorganic solvents known as solvent
system.
The sample is applied on one end of the filter paper and then dipped in the solvent.
The solvent is then allowed to rise by capillary action (ascending chromatography)
or move down by capillary action (descending chromatography).
 THIN LAYER CHROMATOGRAPHY

In TLC a thin layer of adsorbent, such as silica gel is spread uniformly on

a glass plate, which is then activated at high temperature (stationary

phase).

The sample is added as a small spot or band near the edge of the plate.

The plate is then placed in a closed glass container or tank with the lower

edge in and the sample band just above the mobile phase.
 COLUMN CHROMATOGRAPHY

The mobile phase or eluent exits from the column and passes through a
detector that produces an electronic signal.
The resultant graphical display is a chromatogram. The retention time
is the time taken for a solute to exit the column and pass through the
detector and is characteristic for a solute.
Solutes are identified qualitatively by their similar retention times.
 Column chromatography
• It involves two phases.
• Stationary phase and mobile phase ,
• Stationary phase consists of a packed column composed of spherical beads of cellulose,
silica or acrylamide.
• When solution containing proteins or other substances (mobile phase) is made to pass
through column, various proteins depending upon their charge, hydrophilic interactions bind
with the functional groups of packed material.
• Afterwards these proteins are separated from their binding sites or eluted or run after the
other by running salt solution of different concentrations and at different pH values.
• This column chromatography is of two types;
• Hydrophilic interaction chromatography
• Ion exchange chromatography.
 Applications
• In forensic investigations; testing the levels of alcohol, drugs
poisons in body.

• In Food industry; used for quality control, additives preservatives.

• In proteomic study , compounds of complex mixture of proteins are

identified.
GAS CHROMATOGRAPHY

• GC was developed in 1952 to separate fatty acids.

A gaseous mobile phase is used to pass a mixture of volatile solutes

through a column containing the stationary phase which is liquid.

The mobile phase is typically an inert gas like helium, hydrogen or

argon, referred to as carrier gas.

 Gas Chromatography
• It is a type of partition chromatography in which the stationary phase is a liquid and the
mobile phase is a gas.
• The stationary liquid phase is supported by a column of inert material like silica in a long
narrow column.
• The mixture of substances to be separated is made volatile at one end of the column and
the vapours are swept over the column by an inert carrier gas like argon or nitrogen.
• The fractions emerging from the column are detected and quantified by detector .
• Appliction:
This is more suitable for compounds like liquids which remain stable even at high
temperature.
• The stationary phase is a non-volatile liquid coated onto an inert solid

support.

• Sample vapor is moved through the column partially as gas and partially

as liquid.

• Components with higher boiling point will be retained longer in the

liquid phase while the more volatile solute elutes from the column

dissolved in the carrier gas.

 Applications:
This is more suitable for those liquids which
remain stable even at high temperature.

It can be used to detect alcohols in blood.

It can be used to investigate presence or levels of

aromatic compounds, hydrocarbons, pesticides,
herbicides
 Liquid chromatography and HPLC

• Separation by LC is based on the distribution of the solutes between a

liquid mobile phase and a stationary phase.

• When particles of small diameter are used as the stationary -phase

support, the technique is called high performance liquid

chromatography.
 HPLC
• It is a widely used technique for all types of compounds.

• The liquid phase passes through the column under high pressure (1000 times
atmospheric pressure).

• The column may be packed with materials for adsorption, partition and ion exchange.

• The method is therefore based on the same principle as for those types already
described.

• Separation/purification is achieved with better resolution and high speed within

minutes.
• Modern HPLC detectors monitor the elute as it leaves the column, and

produce an electronic signal proportional to the concentration of each

separated component.

• Relatively high pressures are required to pump liquid through efficient

HPLC columns.

• Spectrophotometers that detect absorbance of visible or ultraviolet light

are most commonly used.

• Applications

• In clinical laboratories, HPLC is the most widely used form of LC.

• It is used for measuring amino acids, peptides, proteins, carbohydrates,

lipids, nucleic acids, vitamins, hormones, antibiotics etc.

• This instrument has advantage of high speed, high resolution and

versatility.
 Applications
• It
can be used to check presence of impurities in a
compound/mixture.

• Isolaion and identification of drugs.

• Purification of proteins, herbal extracts for further analysis.

 Gel filtration chromatography
•Mixture of proteins is passed through a porous gel.

•Initially smaller molecules penetrate the gel and flow past

the gel beads while larger molecular weight proteins are
excluded from gel and exit the column first.
•After wards, the smaller ones leave the gel and are
collected.
So, smaller proteins penetrate internal volume of the beads
and are eluted later from the column.
 Applications
• To separate various nucleic acids DNA, RNA and
tRNA.
• To separate biological molecules of different size.

• To determine relative molecular mass.

 Affinity chromatography
• The technique is based on the high affinity of specific proteins
for specific chemical groups.
• e.g.
Co-enzymes can be used to purify enzymes, e.g. NAD is
used to purify dehydrogenase.
• When a solution containing variety of proteins is passed
through column, the protein to be separated gets bound to the
substrate or the co-enzyme from which it is later separated and
obtained in purified form.
 Principle
• Itis based on isolation of target molecules on basis of
specific biological interaction. For example;
• Antigen: antibody

• Lectin: carbohydrate

• Chelated metal ion and histidine peptide

 Applications
• This technique is used to separate and identify amino acids , carbohydrates,
fats and other natural substances.
• It an be used to purify sufficient amounts of particular substances.

• Affinity gel column are used in clinical labs to separate and quantify HbA1c.

• Purifications ofantigens by binding them with specific antibodies.

• Conversely, antibodies can be purified by passing through the column having
specific antigen.
• Detection of substrate.
Affinity chromatography
 Ion Exchange chromatography
• Itis based on separation of compounds based on their net charge.
• Ion exchange chromatography media contain negatively or
positively charged functional groups covalently bound to solid
support, yielding either cation or anion exchanger, respectively.
• Cation exchange chromatography is used when molecule of
interest is positively charged.
• Anion exchange chromatography is used when stationary phase is
positively charged and negatively charged molecules are loaded
to be attracted to it.
 Resins
• Resinsare used in this method which are insoluble synthetic polymers of
two types;
• Cation and anion exchange resins:
• Acidic or cation exchange resins; these contain acidic groups,
carboxylic group, sulphonic acid, phenolic group.
• Basic or anion exchange resins; these contain weakly basic groups like
–NH2 or a strongly basic quaternary ammonium group (-NR3+). These
resins can bind negatively charged groups like hydroxyl, halide, citrate,
sulphate.
 Applications
• An example of use of anion exchangers in medicine is its
administration by mouth to bind gastric HCl in treatment of
peptic ulcer.
• It is used in separation and purification of amino acids,
vitamins and hormones.

• Production of low sodium milk for special dietary needs.

• Removal of excess of Na+ and K+ from body fluids in

congestive heart failure and renal failure , respectively.
 Applications
•It can be used in protein purification.

•Water analysis is also done by this technique.

•Qualitycontrol assays in labs or industries can be

performed using this method.
 Radio immuno Assay
• This technique is extremely sensitive and specific.
• Principle
• It follows antigen antibody complex formation.
• Purified hormone is injected into rabbits, which make antibodies against it. The antibody is
purified.
• A radioactively labelled hormone is allowed to bind with constant amount of antibody.
• A constant amount of antibody is incubated with a fixed amount of radioactively labelled
hormone.
• A certain fraction of radio-labeled hormone binds to the antibody.
• The hormone to be measured that is non radioactively labelled is added to this antigen antibody
complex, this un-labelled hormone or antigen competes with and displaces some of the labelled
hormone from the antibody.
• The fraction of the displaced labeled hormone is measured, it is equal to amount of un-labelled
hormone.
 Applications
• Thismethod can be used to measure amount of peptides including
peptide hormones.

• Detection of different viral antigens.

• Detection of early stage of cancer.

• This sensitive method can be applied to measure levels of hormones

in picogram (10-12 grams).
 ELISA TECHNIQUE

ENZYME LINKED IMMUNOSORBENT ASSAY

 Definitions
Antibodies (also known as immunoglobulins abbreviated as Ig) are
gamma globulin proteins that are found in blood and are used by
the immune system to identify and neutralize foreign objects, such
as bacteria and viruses.
 Cont.
 Antigens

A substance that when introduced into the body

stimulates the production of an antibody

 Immunoassay

A laboratory technique that makes use of the binding between

an antigen and its homologous antibody in order to identify
and quantify the specific antigen or antibody in a sample
Analyte
The sample being analyzed in an immuno-assay. The analyte is either
Antibody or Antigen.

An Antigen…
 May be present naturally in the body like hormones.

 Can be manufactured in special disease status for example human

chorionic gonadotrophin hormone (HCG) which is normally produced
by cells of the placenta in pregnancy is found in the body in some types of
cancer.

 Is not present in the body in normal condition like drugs, allergen, virus
 Types of ELISA technique

1- Competitive ELISA

2- Sandwich ELISA (also called direct ELISA)

3- Indirect ELISA
 ELISA Plate

• 96 well plate
• Made of plastic on which protein can be adsorbed
(bind) easily.
ELISA
• It is most sensitive method as compared to radio-immunoassay.
• Principle
• A specific antibody is fixed to the well of a microtiter plate.
• Thepatient serum is added in the well and incubated for 30 minutes at
37°C
• If the serum contains that specific antigen, it is fixed on the antibody.
• Excess antigen and unwanted proteins are washed away.
• Thenthe enzyme horse raddish peroxidase-labelled antibody is added which
forms a sandwich complex of antibody-antigen-antibody enzyme.

• Enzyme substrate is added and colour is developed.

• Intensity of the colour is measured from which the concentration of an antigen is

calculated.

• Applications

• This
technique is used for the quantification of antigens, antibodies, hormones,
growth factors, tumour markers, cytokines and bacterial antigens.
 ELISA Reader
Plate is read at Appropriate wavelength
(=450 nm) for HRP 405nm for ALP.
 Results

 Afterreading the results the standard curve is drawn.

The concentration is plotted on the X-axis and the
absorbance on the Y-axis.

Absorbance (nm)

Concentration (ng/ml)
Results Cont.

This standard curve is used to determine the

unknown concentration of each sample by
finding the concentration to the absorbance.

Absorbance
(nm)

Concentration (ng/ml)
 Electrophoresis
Principle
• If an electrical current is passed through a protein solution whose pH has been shifted
either above or below it's isoelectric pH, the protein molecule will travel to the
oppositely charged pole/electrode.
• For example, at a pH above the isoelectric point pI, proteins have a negative charge
and therefore move towards that opposite pole or electrode.

• Electrophoresis is of many types;

• SDS PAGE
• PAGE
• Two dimensional gel electrophoresis
• Agarose gel electrophoresis
 SDS-PAGE ELCTOPHORESIS
• It
is a latest technique by the use of sodium dodecyl sulphate SDS-
PAGE.

• SDS has the property of binding with most of the proteins which is
in proportion to the molecular weight or size of the proteins.

•A mixture of proteins is placed in a well, made in the gel which is

subjected to an electric field. The proteins will move depending upon
their molecular weights. The various protein fractions can be
identified by staining with suitable dyes.
 Applications
• It
is used for separation of different protein fractions from a mixture of
proteins.

• This method is also used for separation of lipoproteins or nucleic acids.

• It is used for DNA sequencing.

• Food industry.

• Used for estimation of molecular weight of proteins.

 Spectrophotometry
Principle

This technique is used to measure the amount of light that a sample absorbs.
It works on the principle of Beer’s- Lambert’s law which states that there is a
linear relationship between the absorbance and the concentration of a sample.
Beer’s law
Absorbance of light is directly proportional to concentration of solute.
Lambert's law
Absorbance of light is directly proportional to thickness of material /medium.
 Components
• Light source
• Filter for selecting monochromatic light
• Sample holder or cuvette
• Detector and digital display

• Theinstrument is operated by passing a beam of light through sample

solution and measuring the intensity of light reaching to detector.
 Applications
• This instrument is 1000 times more sensitive than colorimeter.

• Very minute quantities of the substances can be measured.

• This technique is used for clinical diagnosis and in research

laboratories for quantitative estimations of compounds in various
biological fluids. For example, blood glucose, blood urea, serum
cholesterol serum creatinine, serum bilirubin and different
enzymes.
 Polymerase chain reaction PCR

• Repetitive procedure that results in a amplification of specific

DNA sequence.

• It can amplify up to 1,000,000 or more times a segment of DNA.

The reagents required for PCR are as follows:

• Target DNA segment or gene to be amplified.

• Synthetic oligonucleotides

• DNA polymerase enzyme that is also known as Taq polymerase, this is

obtained from a bacterium thermus acquitus that grows in hot springs
and can tolerate temperature upto 90°C.

• Four deoxyribonucleoside triphosphates, i.e. dATP dCTP dTTP and

dCTP.
 Procedure
• The whole procedure is carried out by automated mechanism.

• Firstof all, target DNA segment (double stranded) is selected which acts
as a template for synthesis of new copies of DNA in large quantity.

• DNA is heated or melted gradually to separate its two strands.

• Synthetic oligonucleotides are added to the heated DNA.

• DNA is cooled, that process is known as an annealing.

• Then DNA polymerase is added that will result in

synthesis of daughter strands in 5’-3’ direction.

• DNA is again heated and all the above steps will be

repeated 25 to 30 times.
Applications of PCR
• This method can be used to amplify targeted DNA.
• It is helpful for further DNA sequencing
• It is also used for mutation analysis
• Thistechnique is implied for pre-natal diagnosis of
inherited diseases.
• DNA fingerprinting is also done by this method.
 Dialysis
Dialysis can be defined as a spontaneous separation process of suspended

colloidal particles from dissolved ions or molecules of small dimensions

through a semi permeable membrane.

Most common dialysis membranes are made of cellulose, modified

cellulose or synthetic polymer (cellulose acetate or nitrocellulose).

 Principle
• Itworks on principle of osmosis and filtration of fluid across a
semipermeable membrane with use of dialysate.
Applications
• Used to treat fluid and electrolyte imbalance
• To remove waste products in renal failure
• To purify protein products
• Usedto eliminate small and unwanted molecules such as salt and large
molecules like dyes in solution.
•Dialysis occurs when a sample contained in a
cellulose bag and is immersed into a dialysate
solution.

• During dialysis, equilibrium is achieved between

the sample and dialysate since only small molecules
can pass the cellulose membrane, leaving only larger
particles behind.