Unit–IV: ELECTROPHORETIC AND SPECTROSCOPY TECHNIQUES

ELECTROPHORESIS OF PROTEINS:
Protein electrophoresis is the process of separating samples of proteins based on size.
PAGE (Polyacrylamide Gel Electrophoresis):
Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry,
genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic
acids, according to their electrophoretic mobility.
The most commonly used form of polyacrylamide gel electrophoresis is the Sodium dodecyl suplhate
Polyacrylamide gel electrophoresis (SDS- PAGE) used mostly for the separation of proteins.
SDS PAGE / Sodium Dodecyl Sulphate-Polyacrylamide Gel:
SDS PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for the
separation of proteins based on their molecular weight. It is a technique widely used in forensics, genetics,
biotechnology and molecular biology to separate the protein molecules based on their electrophoretic mobility.
Principle of SDS-PAGE
This technique uses anionic detergent sodium dodecyl sulfate (SDS) which disassociates proteins into their
individual polypeptide subunits and gives a uniform negative charge along each denatured polypeptide. When these
denatured polypeptides are loaded at the cathode end of an electric field, then we get clear bands of proteins arranged
in decreasing order of their molecular mass from the cathode to anode.
Role of SDS in SDS-PAGE
SDS is a detergent present in the SDS-PAGE sample buffer. SDS along with some reducing agents function
to break the disulphide bonds of proteins disrupting the tertiary structure of proteins.

Materials Required
• Power Supplies: It is used to convert the AC current to DC current.
• Gels: These are either prepared in the laboratory or precast gels are purchased from the market.
• Electrophoresis Chambers: The chambers that can fit the SDS-PAGE gels should be used.
• Protein Samples: The protein is diluted using SDS-PAGE sample buffer and boiled for 10 minutes. A
reducing agent such as dithiothreitol or 2-mercaptoethanol is also added to reduce the disulfide linkages to
prevent any tertiary protein folding.
• Running Buffer: The protein samples loaded on the gel are run in SDS-PAGE running buffer.
• Staining and Destaining Buffer: The gel is stained with Coomassie Stain Solution. The gel is then
destained with the destaining solution. Protein bands are then visible under naked eyes.
• Protein Ladder: A reference protein ladder is used to determine the location of the protein of interest,
based on the molecular size.

Protocol of SDS-PAGE
Preparation of the Gel
• All the reagents are combined, except TEMED, for the preparation of gel.
• When the gel is ready to be poured, add TEMED.
• The separating gel is poured in the casting chamber.
• Add butanol before polymerization to remove the unwanted air bubbles present.
 • The comb is inserted in the spaces between the glass plate.
• The polymerized gel is known as the “gel cassette”.
Sample Preparation
• Boil some water in a beaker.
• Add 2-mercaptoethanol to the sample buffer.
• Place the buffer solution in microcentrifuge tubes and add protein sample to it.
• Take MW markers in separate tubes.
• Boil the samples for less than 5 minutes to completely denature the proteins.
Electrophoresis
• The gel cassette is removed from the casting stand and placed in the electrode assembly.
• The electrode assembly is fixed in the clamp stand.
• 1x electrophoresis buffer is poured in the opening of the casting frame to fill the wells of the gel.
• Pipette 30ml of the denatured sample in the well.
• The tank is then covered with a lid and the unit is connected to a power supply.
• The sample is allowed to run at 30mA for about 1 hour.
• The bands are then seen under UV light.

Applications of SDS-PAGE
The applications of SDS-PAGE are as follows:
1. It is used to measure the molecular weight of the molecules.
2. It is used to estimate the size of the protein.
3. Used in peptide mapping
4. It is used to compare the polypeptide composition of different structures.
5. It is used to estimate the purity of the proteins.
6. It is used in Western Blotting and protein ubiquitination.
7. It is used in HIV test to separate the HIV proteins.
8. Analyzing the size and number of polypeptide subunits.
IEF (ISO ELECTRIC FOCUSING) ELECTROPHORESIS
Isoelectric focusing (IEF) is a technique for separating different molecules
by differences in their isoelectric point (pI). It is a type of electrophoresis,
usually performed on proteins in a gel, that takes advantage of the fact that
overall charge on the molecule of interest is a function of the pH of its
surroundings. When an IEF gel is poured a pH gradient is established
A protein that is in a pH region above its isoelectric point (pI) will
be negatively charged and will migrate towards the anode (positive). As it
migrates through a gradient of decreasing pH, however, the protein's overall
charge will increase until the protein reaches the pH region that corresponds
to its pI. At this point it has no net charge and so migration ceases (as there
is no electrical attraction towards either electrode). As a result, the proteins
become focused into sharp stationary bands with each protein positioned at
a point in the pH gradient corresponding to its pI. The technique is capable
of extremely high resolution with proteins differing by a single charge being
fractionated into separate bands.
APPLICATIONS  Widely used for separation and identification of serum proteins.  Used in food and
agricultural industries, forensic and human genetics laboratories.  Used in enzymology, immunology and
membrane biochemistry.  2D Gel electrophoresis is an application of IEF. Protein is first separated based on pI and
then based on molecular weight using SDS-PAGE.

Advantages

Proteins that by as little as pH units can be separated. As spreading of bands is minimized due to application
of the applied field and the pH gradient, high resolution can be achieved. IEF is a powerful analytical tool for the
separation of proteins. Performing IEF is easier because the placement of sample application is not important.

Disadvantages

Carrier ampholytes are generally used in high concentration, a high voltage (up to 2000v) is necessary. As a
result the electrophoretic matrix must be cooled which sometimes makes it difficult. Limited stability of solutions.
Lot-to-lot inconsistency. Inadequate purity for application as a standard. high volume of rehydration solution needs.
cassette leaking due to urea and detergent. rehydration loading of different sample is not possible.

2D GEL ELECTROPHORESIS

Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel

electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two
dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
• Analysis of sample by one-dimensional electrophoresis is the most common form of protein gel
electrophoresis.
• For separation and analysis of hundreds to thousands of proteins in one gel, a powerful electrophoretic
method called two-dimensional gel electrophoresis is used.
• 2D gel electrophoresis separates a mixture of proteins according to two properties, one in each dimension.
• The first dimensions involve the separation based on native isoelectric point (pI), using form of
electrophoresis called isoelectric focusing (IEF).
• Second dimensions separate mass using SDS-PAGE.
• This technique provides highest resolution for the protein analysis.

Visualization After electrophoresis the gel is stained to visualize the separated proteins. Commonly used
stained are Coomassie Brilliant Blue or SYPRO RUBY or Sliver Stain, different proteins will appear as distinct spot
within gel.
Application To protein can be separated in pure form from spots which can be quantified and also analyzed by
MS. To study of gene products at molecular level. To study proteomics. Provides important information
correlating the absence or presence of individual protein characteristic of specific clinical conditions.

Advantages of 2D gel electrophoresis

Resolution: proteins from 10 4 -10 6 a sample. Preset conditions ( pH ranges, size of the gel, staining methods etc)
can be changed to increase resolution. Delivers a map of intact protein that can be stored and analyzed at will. One
of the core technology of proteomics

Disadvantages This technique include a large amount of sample handling, less reproducibility. It is also not
automated for high throughput analysis. 2D- PAGE has limited dynamic range. Difficulty to separate low
abundance proteins, acidic and basic proteins, very large and very small proteins and hydrophobic proteins.

DETECTION, ESTIMATION AND RECOVERY OF PROTEIN IN GELS

Detection of protein: The most commonly used general protein stain for detecting protein on gels is the sulfated
try methylamine dye Coomassie Brilliant Blue R-250 (CBB). The name Coomassie was adopted at the end of the
19th century as a trade name by the Blackley-based dye manufacturer Levinstein Ltd, in marketing a range of acid
wool dyes. In 1896 during the Fourth Anglo-Ashanti War, British forces had occupied the town of Coomassie
(modern-day Kumasi in Ghana). Coomassie Brilliant Blue R-250 was first used to visualise proteins in 1964 by
Fazekas de St. Groth and colleagues. Protein samples were separated electrophoretically on a cellulose acetate sheet.

Preperation: Staining is usually carried out using 0.1% (W/V) CBB in methanol: water: glacial acetic acid
(45:45:10). This acid methanol mixture acts as denaturant to precipitate or fix the protein gel, which prevents the
protein from being washed out whilst it is being stained. Staining of most gels accomplished in about 2 hour
destaining, usually overnight is achieved by gentle agitation in the same acid- methanol solution, but in the absence
of the dye. The coomassie stain is highly sensitive; a very weakly staining on band on a polyacrylamide gel would
corresponds to about 0.1µg (100ng) of protein .

Although coomassie blue stain is highly sensitive, many workers require greater sensitivity such as provided
by silver staining. Silver stains are based either on techniques developed for histology or on methods based on
the photographic process. In either case silver ions (Ag˖) are reduced to metollic silver on the protein, where silver
is deposite to give a black or brown bands. Silver may commence immediately after electrophoresis, or
alternatively, after staining with CBB. The silver stain is atleast 100 times more sensitive than CBB, detecting
proteins down to 1ng amounts.

Estimation of Protein in gels: Quantitative analysis (i.e. measurement of the relative amounts of different
proteins in a sample) can be achieved by Scanning Densitometry. A number of commercial scanning
densitometers are available, and work by passing the stained gel track over a beam of light and measuring the
transmitted light; standard office desktop scanners can also be used for this purpose. A graphics presentation of
protein zones (peak of absorbance) against migration distance is produced, and peak areas can be calculated to obtain
quantitative data (e.g. using the software image) However, such data must interpreted with caution because there
is only a limited range of protein concentrations over which there is a linear relationship between absorption and
concentration.

More recently gel documentation systems have been developed, which are replacing scanning densitometers.
Such benchtop system comprise a video imaging unit (computer linked) attached to a small ‘darkroom’ unit that
is fitted with a choice of white or ultraviolet light (transilluminator). Gel images can be stored on the computer,
enhanced accordingly and printed as required on a printer, thus eliminating the need for wet developing in a purpose-
built dark room, as is the case for traditionally photography.

Recovery of protein: Although gel electrophoresis is used generally as an analytical tool, it can be utilized to
separate proteins in a gel to achieve protein purification. Protein bands can be cut out of protein blots and sequence
data obtained by subjecting the blot to mass spectrometric analysis. Stained protein bands can be cut out of protein
gels and the protein recovered by electrophoresis of the protein out of the gel piece (electroelution). A number of
diffenent designs of electroelution cells are commercially avalible, but perhaps the easiest method is to seal the gel
piece in buffer in a dialysis sac and place the sac in buffer between two electrodes.

Protein will migrate out of the gel piece towards the appropriate electrode, but will be retained by the dialysis sac.
After electro elution, the current is reversed for a few seconds to drive of any protein that has absorbed to the wall
of the dialysis sac and then the protein solution within the sac is recovered.

Proteins are extracted from gels by several methods. These include dissolution of the gel matrix, passive diffusion,
and electrophoretic elution.

AGAROSE GEL ELECTROPHORESIS

Agarose gel electrophoresis is a procedure used to separate biological molecules by size. The separation of these
molecules is achieved by placing them in a gel with small pores and creating an electric field across the gel. The
molecules will move faster or slower based on their size and electric charge.
• Agarose gel the supporting media in the electrophoresis.
• For the electrophoresis of DNA, RNA and Protein agrose gel is used.

Principle
In the agrose gel electrophoresis the potential difference is applied across the electrodes in a horizontal
electrophoretic tank containing agarose gel and biomolecules (such as nucleic acid or proteins) is loaded, then
molecules migrated to their respective electrodes. The rate of migration of charged particles depends on the size,
shape, molecular mass etc.
In this process, larger molecules have difficulty in moving through the pore size of the supporting media,
whereas the smaller molecules has more mobility through it. The bands of protein or nucleic acid is visualized by
using intercalating dye, i.e., ethidium bromide (Etbr), they are visualized by fluorescence when illuminated with
ultraviolet lights.

Requirement/ instrumentation:
• An electrophoretic unit,
• A power supply,
• Gel casting trays
 • Combs
• Agarose gel or media
Electrophoresis buffer
Composition and ionic strength of electrophoresis buffer is most important factor for the separation of nucleic acids
(DNA or RNA).
Most routinely used buffers are:
• TAE- (Tris-acetate-EDTA), it has lower buffering capacity and generally used to separate larger nucleic acid
fragments (>12kb).
• TBE- (Tris-borate-EDTA), it has high buffering capacity and higher ionic strength and generally used for
the separation of low molecular weight compound (<1kb).
• Loading buffer: Nucleic acid is before loading on to a gel is first mixed with the gel loading buffer, which
usually consists of:-
• Salts: It creates environment with favorable ionic strength and pH of the sample, e.g., Tris-HCl.
• Metal chelator: It prevents nucleases to degrade the nucleic acid such as EDTA.
• Loading dyes: It provides color for tracking and easy monitoring of sample. Such as, bromophenol blue,
xylene cyanol.
• Transilluminator: (An ultraviolet light box), which is used to visualize bands in gels.
Applications
• In molecular genetic diagnosis or genetic fingerprinting for analysis of PCR products.
• For the estimation of size of DNA molecule.
• In the separation of restricted DNA and RNA.
• In addition to providing an excellent medium for fragment size analyses, agarose gels allow purification of
DNA fragments.

Gel Electrophoresis Steps

The broad steps involved in a common DNA gel electrophoresis protocol:

1. Preparing the samples for running

The DNA is isolated and preprocessed (e.g. PCR, enzymatic digestion) and made up in solution with some basic
blue dye to help visualize the movement of the sample through the gel.

2. An agarose TAE gel solution is prepared TAE buffer provides a source of ions for setting up the electric field
during electrophoresis. The weight-to-volume concentration of agarose in TAE buffer is used to prepare the solution.
For example, if a 1% agarose gel is required, 1g of agarose is added to 100mL of TAE. The agarose percentage used
is determined by how big or small the DNA is expected to be. If one is looking at separating a pool of smaller size
DNA bands (<500bp), a higher percentage agarose gel (>1%) is prepared. The higher percentage of agarose creates
a denser sieve to increase the separation of small DNA length differences. The agarose-TAE solution is heated to
dissolve the agarose.
3. Casting the gel The agarose TAE solution is poured into a casting tray that, once the gel solution has cooled
down and solidified, creates a gel slab with a row of wells at the top.

4. Setting up the electrophoresis chamber The solid gel is placed into a chamber filled with TAE buffer. The gel
is positioned so that the chamber wells are closest to the negative electrode of the chamber.

5. Loading the gel The gel chamber wells are loaded with the DNA samples and usually, a DNA ladder is also
loaded as reference for sizes.

6. Electrophoresis The negative and positive leads are connected to the chamber and to a power supply where the
voltage is set. Turning on the power supply sets up the electric field and the negatively charged DNA samples will
start to migrate through the gel and away from the negative electrode towards the positive.

7. Stopping electrophoresis and visualizing the DNA Once the blue dye in the DNA samples has migrated through
the gel far enough, the power supply is turned off and the gel is removed and placed into an ethidium bromide
solution. Ethidium bromide intercalates between DNA and is visible in UV light. Sometimes ethidium bromide is
added directly to the agarose gel solution in step 2. The ethidium bromide stained gel is then exposed to UV light
and a picture is taken. DNA bands are visualized in from each lane corresponding to a chamber well. The DNA
ladder that was loaded is also visualized and the length of the DNA bands can be estimated.

SANGER’S METHOD OF GENE SEQUENCING

▪ Sanger’s method of gene sequencing is also known as dideoxy chain termination method. It generates nested
set of labelled fragments from a template strand of DNA to be sequenced by replicating that template strand
and interrupting the replication process at one of the four bases.
▪ Four different reaction mixtures are produced that terminates in A. T. G or C respectively.

Principle

▪ A DNA primer is attached by hybridization to the template strand and deoxynucleosides triphosphates
(dNTPPs) are sequentially added to the primer strand by DNA polymerase.
▪ The primer is designed for the known sequences at 3’ end of the template strand.
▪ M13 sequences is generally attached to 3’ end and the primer of this M13 is made.
▪ The reaction mixture also contains dideoxynucleoside triphosphate (ddNTPs) along with usual dNTPs.
▪ If during replication ddNTPs is incorporated instead of usual dNTPs in the growing DNA strand then the
replication stops at that nucleotide.
▪ The ddNTPs are analogue of dNTPs
▪ ddNTPs lacks hydroxyl group (-OH) at c3 of ribose sugar, so it cannot make phosphodiester bond with nest
nucleotide, thus terminates the nucleotide chain
 ▪ Respective ddNTPs of dNTPs terminates chain at their respective site. For example, ddATP terminates at A
site. Similarly, ddCTP, ddGTP and ddTTP terminates at C, G and T site respectively.

Procedure

1. Template preparation:

▪ Copies of template strand to be sequenced must be prepared with short known sequences at 3’ end of the template strand.
▪ A DNA primere is essential to initiate replication of template, so primer preparation of known sequences at 3’end is always
required.
▪ For this purpose, a single stranded cloning vector M13 is flanked with template strand at 3’end which serves as binding site
for primer.
2. Generation of nested set of labelled fragments:
▪ Copies of each template is divided into four batches and each batch is used for different replication reaction.
▪ Copies of standard primer and DNA polymerase I are used in all four batches.
▪ To synthesize fragments that terminates at A, ddATP is added to the reaction mixture on batch I along with dATP, dTTP,
dCTP and dGTP, standard primer and DNA polymerase I.
▪ Similarly, to generate, all fragments that terminates at C, G and T, the respective ddNTPs ie ddCTP, ddGTP and ddTTP are
added respectively to different reaction mixture on different batch along with usual dNTPs.

3. Electrophoresis and gel reading:

▪ The reaction mixture from four batches are loaded into four different well on polyacrylamide gel and
electrophoresed.
▪ The autoradiogram of the gel is read to determine the order of bases of complementary strand to that of
template strand.
▪ The band of shortest fragments are at the bottom of autoradiogram so that the sequences of complementary
strand is read from bottom to top.
PULSE FIELD GEL ELECTROPHORESIS (PFGE)
• Conventional agarose gel electrophoresis cannot separate linear double stranded DNA molecules that have
radius of gyration, which is larger than the pore size of the gel.
• However, with certain changes in the orientation of electric field with respect to the gel, large DNA fragments
can be resolved.
• It is used to separate large DNA molecule by applying gel matrix as electric field that periodically changes
direction.
• This technique is invented by Schwartz and Cantor in 1984.
 • DNA fragments up to 10 mb can be separated by this technique.

Principle
As opposed to the continuous unidirectional electric fields applied in conventional gel electrophoresis,
pulsed-field gel electrophoresis uses pulsed, alternating, orthogonal electric fields. When such a field is applied to a
gel, large DNA molecules become trapped into their reptation tubes every time the direction of the electric field is
changed.
These molecules remain immobile till they reorient t themselves along the direction of the new electric field.
It is here that different DNA molecules adapt a behavior consonant with their respective sizes; large DNA molecules
take a longer time to reorient themselves and are consequently retarded more in the new electric field as compared
to the smaller DNA molecules.
Thus, all those molecules of DNA whose reorientation times are less than the period of the electric pulse can be
fractionated in a size dependent manner. Factors, which are of extreme importance for determining the limit of
resolution of pulsed-field gel electrophoresis are given below:
1. The absolute periods of the electric pulses.
2. The angles at which the two electric fields are applied to the gel.
3. The relative field strengths of the two electric fields and the degree of uniformity of the two electric fields.
4. The ratio of the periods of the electric pulses employed to generate the two electric fields.
Instrumentation
• The original apparatus used pulsed electric fields or perpendicular orientations and linear electrodes.
Applications
•It is used for the separation of DNA more than 10 mb.
• It generated stable and reproducible DNA restriction patterns.
• This technique applied to the sub-typing of many pathogenic bacteria and has high concordance with
epidemiological relatedness.
• This technique can be applied as a universal generic method for sub-typing of bacteria.
ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA)

The electrophoretic mobility shift assay (EMSA), or gel shift assay is a simple and rapid method to detect
protein complexes with nucleic acids. EMSA originally used widely in the study of sequence-specific DNA-binding
proteins such as transcription factors, has been further developed to investigate DNA-protein interactions, RNA-
protein interactions, and even DNA-RNA interactions. It is also applied to qualify and quantify proteins that
specifically bind to given nucleotides, enabling to accommodate a wide range of binding conditions.

EMSA based on the principle that the rate of migration of the complex of DNA and protein is slower than
single DNA fragment or double stranded oligonucleotide during a nondenaturing polyacrylamide gel. Besides, the
kinetic analysis of EMSA is another basic theory to underlying the method. The protein (P), binding to a unique site
on a DNA (D), will form a complex PD, in equilibrium with the free components:

Where ka is the rate of association and kd is the rate of dissociation. When there is a strong interaction between
protein and DNA, Ka>Kd, a distinct band (PD) is observed. However, because of the dissociation occurs during
electrophoresis, a faint smear would also show between the two major bands. If a single DNA molecule has multiple
binding sites for an individual protein, there will be multiple complexes formed, and we could observe many bands.

Purified proteins, nuclear or cell extract preparations co-incubate with 32P end-labeled DNA fragment containing
the putative protein binding site. Then the reaction products are analyzed on a nondenaturing polyacrylamide gel.
The specific binding-protein was determined by competition experiments. Buffers with high ionic strength during
electrophoresis can produce electrophoretic bands that are more vivid than the buffer profile with low ionic strength.
However, there are some DNA in buffers with high ionic strength cannot interact with protein. Therefore, for each
protein-DNA interaction, both buffer systems should be tested.

EMSA is simple to perform, thus it could be used for a wide range of binding conditions. The assay is also highly
sensitive within small protein and nucleic acid concentrations. And ranges of nucleic acid sizes and structures are
suitable for the assay. Additionally, the EMSA assay words well for both purified proteins and crude cell extracts.

But there are also limitations on EMSA assay. Firstly, weak interaction or rapid dissociation during electrophoresis
can prevent detection of complexes brand. Secondly, electrophoretic mobility of a protein-nucleic acid complex
depends on many factors other than the size of the protein. Thus we could not measure the protein directly by
observing the gel. Thirdly, the result of the assay provides little direct information on the location of the nucleic acid
sequences that binding the protein. Finally, the time resolution of the current assay is defined by the interval required
for manual solution handling.

Procedure:

1. Probe Labeling

1. Select restriction enzymes that produce the shortest DNA fragment containing the sequence of interest.
2. Run a sample on an agarose gel to check the digestion is complete.
3. Add CIP and 10×CIP buffer directly to the digestion reaction mix and fill with H2O. Incubate at 37°C for 90
min.
4. Incubate the reaction mix at 70°C for 10 min to eliminating and inactivating CIP.
5. Precipitate DNA on dry ice for 30min with1/10 volume of NaOAc 3 M pH 5.2 and 2 volumes of cold 90%
ethanol.
6. Centrifuge for 15 min at 4°C.
7. Resuspend DNA in 33 mL of H2O and add 5 mL of 10×kinase buffer, 2 mL of T4 polynucleotide kinase, and
100 mCiof [g-32P] ATP. Mix and incubate at 37°C for 2 h.
8. If needed, precipitate DNA and digest the remainder with the second restriction enzyme following the same
procedure.
2. Probe Isolation

1. Clean and dry the polyacrylamide gel apparatus and its accessories.
2. Prepare the 6% polyacrylamide gel.
3. Mount the gel in the electrophoresis tank and fill the chamber with 1× TBE.
4. Add 6×loading buffer to the digested sample and load into two wells. Migration should be stopped when
bromophenol blue reaches two-third of the gel length.
5. Under radioactive protection, disassemble the apparatus. Mark the gel and labeled bands in a dark room. Using
a razor blade cut the bands interested.
6. Place the acrylamide fragment in a dialysis tubing and add 1 mL of 1× TBE, place the dialysis tubing in a
standard electrophoresis tank for agarose gel. Run for 15min.
7. Using a Pasteur pipette, transfer the labeled probe-containing TBE from the dialysis tubing into microcentrifuge
tubes.
8. Precipitate DNA on dry ice for 30min with1/10 volume of NaOAc 3 M pH 5.2 and 2 volumes of cold 90%
ethanol. Centrifuge for 15 min at 4°C.
9. Resuspend labeled DNA in order to obtain 30,000 cpm/mL.
3. EMSA

1. Clean and dry the polyacrylamide gel apparatus and its accessories.
2. Prepare the 5-15% polyacrylamide gel according the binding protein.
3. Pre-electrophoresis at approximately 10 V cm–1 of gel length. Reduce this voltage if gel heating is evident.
4. Prepare samples and equilibrate for 30 min at 20 °C ± 1°C
5. Add 6×loading buffer to the digested sample and load into two wells. Migration should be stopped when
bromophenol blue reaches two-third of the gel length.
6. Under radioactive protection, disassemble the apparatus, leaving the gel adherent to one of the plates.
7. Gel autoradiography in dark room. Place film or phosphor screen in an exposure cassette. Place the wrapped gel
and plate assembly in the cassette, with gel-side toward film or screen. Close the cassette. Expose film or screen
at 4 °C for intervals up to 24 h, and at –80 °C for longer intervals.
Sample Analysis

Example 1:

Analysis: Protein B binds to DNA and protein A does not interact with DNA.

Example 2
Analysis: Protein A binds to DNA. Protein B binds with DNA if there is protein A. The upper band in column four
presents DNA+A+B. Protein B does not interact with DNA independently.

Advantages:

➢ qualitatively detection of interaction

➢ low cost: no special equipment needed, low amounts of biomolecules

Disadvantages:

➢ No quantitative data (in the best rough estimation of KD)

➢ Interacting biomolecules must have different electrophoretic mobilities

BLOTTING

Different blotting techniques are used to identify unique proteins and nucleic acid sequences. Southern,
northern, and western blot protocols are similar, and begin with electrophoretic separation of protein and nucleic
acid fragments on a gel, which are then transferred to a membrane (nitrocellulose membrane, polyvinylidene
difluoride (PVDF) membrane, etc.) where they are immobilized. This enables radiolabeled or enzymatically labeled
antibody or DNA probes to bind the immobilized target, and the molecules of interest may then be visualized with
various methods. Blotting techniques are selected based on the target molecule: DNA, RNA, or protein.

SOUTHERN BLOT

Southern blots are used to determine the identity, size, and abundance of specific DNA sequences. The
southern blot protocol begins with DNA extraction from the cells or tissues, which is then enzymatically digested
to produce DNA fragments. The fragments are separated by size on an agarose or polyacrylamide gel via
electrophoresis. Smaller fragments will migrate farther on the gel than larger ones. Following electrophoresis, the
DNA on the gel is transferred to a nylon membrane. The membrane is incubated with a nucleic acid probe that has
a sequence homologous to the target sequence and is labeled with radioactivity, fluorescent dye, or an enzyme
capable of generating a chemiluminescent signal. Hybridization of complementary sequences occurs during
incubation, and the unhybridized probe is removed by washing with buffer. The fully hybridized labeled probe
molecules will remain bound to the blot. Detection methods differ based on the probe label; radiolabeled probes are
visualized with X-ray film or phosphor imaging, and enzymatically labeled probes are visualized with
chemiluminescent substrate.

Southern blot protocol

 1. DNA isolation
2. Restriction digestion: digest the DNA with a restriction enzyme, and if necessary, concentrate digested DNA.
3. Gel electrophoresis: prepare an agarose gel and either TAE or TBE buffer (buffer selection will depend on
the duration of the run and the size of the DNA fragments). Load samples into wells and include a DNA
molecular weight marker. Run the gel.
4. Transfer:
o Place the gel in a container with denaturing solution, and wash twice for 15 minutes on a shaker.
o Rinse with water, then wash with neutralization solution.
o During the previous step, begin to prepare Whatman paper and nylon membrane for the transfer.
o Assemble the transfer apparatus with the membrane, Whatman paper, and gel and transfer in SSC or
SSPE buffer.
o When transfer is complete, cross-link DNA in a cross-linker, then rinse the membrane.
5. Pre-hybridization (blocking):
o Blocking reduces non-specific binding to the membrane. Prepare the pre-hybridization solution and
add sample DNA. Remove the blot from the cross-linker, add the pre-hybridization solution and
incubate.
6. Hybridization:
o Prepare the probe mixture (a complementary DNA strand) and buffer.
o Remove the pre-hybridization solution and incubate the blot with the probe (incubation times will
vary depending on the application).
o Following incubation, perform a low-stringency wash followed by a high-stringency wash to refine
the DNA.
7. Probe detection:
o Rinse the membrane, transfer to a container with blocking solution and incubate.
o Discard blocking solution, replace with antibody solution and incubate.
o Discard antibody solution, wash the membrane.
8. Follow manufacturer directions for chemiluminescent detection.

Applications of Southern Blot:

➢ identification of a single gene in a pool of DNA fragments.

➢ gene mapping.
➢ analysis of genetic patterns of DNA.
➢ detection of specific DNA sequences in a genome.
➢ study of gene deletions, duplications, and mutations that cause various diseases.

NORTHERN BLOT

Northern blots are used to determine the identity, size, and abundance of specific RNA sequences. Northern
blot protocols begin with RNA isolation, and separation techniques vary depending on RNA size. Large RNAs are
separated by electrophoresis on a formaldehyde agarose gel or glyoxal agarose gel, which prevents normal base
paring and maintains RNA in a denatured state. Small RNAs are separated on a denaturing (urea) polyacrylamide
gel. The RNA is then transferred from the gel to a nylon membrane which is then incubated with a radioactively or
non-isotopically labeled RNA, DNA, or oligodeoxynucleotide probe. The unhybridized probe is removed by
washing with buffer. Radiolabeled probes are visualized with X-ray film, and enzymatically labeled probes are
visualized with chemiluminescence.

Northern blot protocol

1. RNA isolation
2. Electrophoresis:
o For a formaldehyde agarose gel: prepare the gel and insert the gel tray into the apparatus. Fill with
MOPS buffer, load the samples and include a molecular weight marker. Run the gel, then trim the
gel prior to blotting.
o For a glyoxal agarose gel: prepare the gel and insert the gel tray into the apparatus. Fill with MOPS
buffer, prepare samples and load into wells along with RNA ladder.
 o For a denaturing polyacrylamide gel: cast the gel, and mount it in the electrophoresis unit. Prepare
samples, load into the gel, and run with TBE running buffer.
3. Transfer:
o For a formaldehyde agarose gel or glyoxal agarose gel: wash the gel in SSC, then assemble the
transfer unit with the gel, filter paper, and nylon membrane. When transfer is complete, place the
membrane in a UV cross-linker.
o For a denaturing polyacrylamide gel: assemble the transfer unit including gel, filter paper, and nylon
membrane ensuring they are flooded with TBE. When transfer is complete, place the membrane in a
UV cross-linker to fix the RNA to the membrane.
4. Pre-hybridization (blocking):
o Pre-hybridize the membrane in hybridization solution.
5. Hybridization:
o Add probe to the hybridization solution and incubate.
o Wash the membrane in low-stringency washes to remove hybridization solution and unhybridized
probe, and high-stringency washes to remove partially hybridized molecules.
o Follow manufacturer directions for chemiluminescent detection.

Applications of Northern Blot

➢ The technique can be used for the identification and separation of RNA fragments collected from different
biological sources.
➢ Northern blotting is used as a sensitive test for the detection of transcription of DNA fragments that are to
be used as a probe in Southern Blotting.
➢ It also allows the detection and quantification of specific mRNAs from different tissues and different living
organisms.
➢ Northern blotting is used as a tool for gene expression studies related to overexpression of cancer-causing
genes, and gene expression during transplant rejects.
➢ Northern blotting has been used as a molecular tool for the diagnosis of diseases like Crohn’s disease.
➢ The process is used as a method for the detection of viral microRNAs that play important roles in viral
infection.

WESTERN BLOT

Western blots are used to determine the identity, size, and abundance of specific proteins within a sample.
The western blot protocol begins with sample lysate preparation from tissue or cell culture and separation on a
polyacrylamide gel via electrophoresis. The separated proteins are then transferred to a nitrocellulose or
polyvinylidene difluoride (PVDF) membrane. The membrane is incubated with a blocking agent to prevent
nonspecific binding, followed by incubation with a primary antibody to bind the protein of interest. There are two
detection methods, direct and indirect. Direct detection (Figure 2) relies on a labeled primary antibody, whereas
indirect detection requires a primary antibody directed against the target protein, and a secondary antibody directed
against the immunoglobin class or subclass of the primary antibody’s species (Figure 3). Visualization methods
include colorimetric assays in which a colored precipitate is produced, chemiluminescence, and fluorescence.
Western blot protocol

Step I: Extraction of Protein

▪ Cell lysate is most common sample for western blotting.
▪ Protein is extracted from cell by mechanical or chemical lysis of cell. This step is also known as tissue
preparation.
▪ To prevent denaturing of protein protease inhibitor is used.
▪ The concentration of protein is determined by spectroscopy.
▪ When sufficient amount of protein sample is obtained, it is diluted in loading buffer containing glycerol which
helps to sink the sample in well.
▪ Tracking dye (bromothymol blue) is also added in sample to monitor the movement of proteins.
Step II: Gel electrophoresis
▪ The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate- poly-acrylamide gel electrophoresis.
▪ The proteins are separated on the basis of electric charge, isoelectric point, molecular weight, or combination
of these all.
▪ The small size protein moves faster than large size protein.
▪ Protein are negatively charged, so they move toward positive (anode) pole as electric current is applied.
Step III: Blotting
▪ The nitrocellulose membrane is placed on the gel. The separated protein from gel get transferred to
nitrocellulose paper by capillary action. This type of blotting is time consuming and may take 1-2 days
▪ For fast and more efficient transfer of desired protein from the gel to nitrocellulose paper electro-blotting can
be used.
▪ In electro-blotting nitrocellulose membrane is sandwich between gel and cassette of filter paper and then
electric current is passed through the gel causing transfer of protein to the membrane.
Step IV: Blocking
▪ Blocking is very important step in western blotting.
▪ Antibodies are also protein so they are likely to bind the nitrocellulose paper. So before adding the primary
antibody the membrane is non-specifically saturated or masked by using casein or Bovine serum albumin
(BSA).
Step V: Treatment with Primary Antibody
▪ The primary antibody (1° Ab) is specific to desired protein so it form Ag-Ab complex
Step VI: Treatment with secondary antibody
▪ The secondary antibody is enzyme labelled. For eg. alkaline phosphatase or Horseradish peroxidase (HRP) is
labelled with secondary antibody.
▪ Secondary antibody (2° Ab) is antibody against primary antibody (anti-antibody) so it can bind with Ag-Ab
complex.
Step VII: Treatment with suitable substrate
▪ To visualize the enzyme action, the reaction mixture is incubated with specific substrate.
▪ The enzyme convert the substrate to give visible colored product, so band of color can be visualized in the
membrane.
▪ Western blotting is also a quantitative test to determine the amount of protein in sample.
Application:
➢ To determine the size and amount of protein in given sample.
➢ Disease diagnosis: detects antibody against virus or bacteria in serum.
➢ Western blotting technique is the confirmatory test for HIV. It detects anti HIV antibody in patient’s serum.
➢ Useful to detect defective proteins. For eg Prions disease.
➢ Definitive test for Creutzfeldt-Jacob disease, Lyme disease, Hepatitis B and Herpes
SOUTHWESTERN BLOT: The southwestern blot, is a lab technique that involves identifying as well as
characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes. Determination of
molecular weight of proteins binding to DNA is also made possible by the technique.
Combines features of Southern and Western blotting techniques.
For rapid characterization of both DNA binding proteins and their specific sites on genomic DNA.
Involves identifying and characterizing DNA-binding proteins (proteins that bind to DNA) by their ability
to bind to a specific oligonucleotide probes.
Identification of protein factors that bind to genes to turn them on or off is therefore important in investigating
gene functions.
Primary use is for research, not clinical applications.
Southwestern Blot Procedure – Separate proteins using SDS-PAGE – Renatured by removing SDS in presence
of urea – Transfer to membrane – Genomic DNA of interest is digested by restriction enzymes and labeled – Then
added to separated proteins.

SPECTROSCOPY TECHNIQUES:
Mass Spectrometry (MS)
➢ Mass Spectrometry (MS) is an analytical chemistry technique that helps identify the amount and type of
chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions.
➢ In this instrumental technique, the sample is converted to rapidly moving positive ions by electron
bombardment and charged particles are separated according to their masses.
➢ A mass spectrum is a plot of relative abundance against the ratio of mass/charge (m/e).
➢ These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles
and of molecules, and to elucidate the chemical structures of molecules and other chemical compounds.
Principle:

➢ In this technique, molecules are bombarded with a beam of energetic electrons.

➢ The molecules are ionized and broken up into many fragments, some of which are positive ions. Each kind
of ion has a particular ratio of mass to charge, i.e. m/e ratio (value).
➢ For most ions, the charge is one, and thus, the m/e ratio is simply the molecular mass of the ion.
➢ The ions pass through magnetic and electric fields to reach the detector where they are detected and signals
are recorded to give mass spectra.
Instrumentation
A. Sample Inlet
• A sample stored in the large reservoir from which molecules reach the ionization chamber at low pressure in a
steady stream by a pinhole called “Molecular leak”.
B. Ionization
• Atoms are ionized by knocking one or more electrons off to give positive ions by bombardment with a stream
of electrons. Most of the positive ions formed will carry a charge of +1.
• Ionization can be achieved by:
• Electron Ionization (EI-MS)
• Chemical Ionization (CI-MS)
• Desorption Technique (FAB)
C. Acceleration
• Ions are accelerated so that they all have the same kinetic energy.
• Positive ions pass through 3 slits with voltage in decreasing order.
• Middle slit carries intermediate and finals at zero volts.
D. Deflection
• Ions are deflected by a magnetic field due to differences in their masses.
• The lighter the mass, the more they are deflected.
• It also depends upon the no. of +ve charge an ion is carrying; the more +ve charge, the more it will be
deflected.
E. Detection
• The beam of ions passing through the mass analyzer is detected by a detector on the basis of the m/e ratio.
• When an ion hits the metal box, the charge is neutralized by an electron jumping from the metal onto the
ion.
• Types of analyzers:
• Magnetic sector mass analyzers
• Double focussing analyzers
• Quadrupole mass analysers
• Time of Flight analyzers (TOF)
• Ion trap analyzer
• Ion cyclotron analyser
Applications of Mass Spectrometry (MS)
• Environmental monitoring and analysis (soil, water, and air pollutants, water quality, etc.)
• Geochemistry – age determination, soil, and rock composition, oil and gas surveying
• Chemical and Petrochemical industry – Quality control
• Identify structures of biomolecules, such as carbohydrates, nucleic acids
• Sequence biopolymers such as proteins and oligosaccharides
• Determination of the molecular mass of peptides, proteins, and oligonucleotides.
• Monitoring gases in patients’ breath during surgery.
• Identification of drug abuse and metabolites of drugs of abuse in blood, urine, and saliva.
• Analyses of aerosol particles.
• Determination of pesticides residues in food.

MASS ANALYZER
Mass analyzer is also called ion separator. Mass analyzer is the Heart of the mass spectrometer that
takes ionized masses and separates them based on charge to mass ratios.
Mass analyzer must posses the following characteristics.
It should have a high resolution. It must have a high rate of transmission of ions.
Types of analyzers:
• Magnetic sector mass analyzers
• Double focussing analyzers
• Quadrupole mass analysers
• Time of Flight analyzers (TOF)
• Ion trap analyzer
• Ion cyclotron analyser
Magnetic sector mass analyzer
 In Magnetic Sector Mass Analyzer, ions are accelerated so that they have the same kinetic energy. All the
ions are accelerated into a focused beam. And then the ions are deflected by the magnetic field according to masses
of ions. The lighter ions have more deflection than the heavier ones. The amount of deflection depends on the
number of the positive charges. When similar ions pass through the magnetic field, they all will be deflected to the
same degree and will all follow the same trajectory path. Those ions which are not selected will collide with either
side of the flight tube wall or will not pass through the slit to the detector.

Time of flight (TOF)

A time of flight analyzer consists of a pulsed ion source, an accelerating grid, a field-free flight tube, and a
detector. The flight time needed by the ions with a particular mass to charge, accelerated by a potential voltage, to
reach the detector placed at a distance, can be calculated from a formula.
Pulsing of the ion source is required to avoid the simultaneous arrival of ions of different m/z at the detector
At high masses, not all the ions of the same m/z values reach their ideal velocities. To fix this problem, often a
reflection which consists of a series of ring electrodes with high voltage is added to the end of the flight tube.
Because of the high voltage, an ion is reflected in the opposite direction, when it into the reflection. For the ions of
same m/z value, faster ions travel further than the slower ones into the reflections. In this way, both the slow and
fast ions of the same m/z value reach the detector at the same time. The reflection increases resolution by
narrowing the broadband range of flight times for a single m/z value.

Quadrupole

The quadrupole consists of four parallel metal rods and each opposing rod pair is connected together electrically.
One pair of raids is applied with a radio frequency (RF) voltage while another one is applied with a direct current
(DC) voltage. At a given DC and RF combination, only the ions of a particular m/z show a stable trajectory and can
be transmitted to the detector, while other ions with unstable trajectories don’t pass the road, because the amplitude
of their oscillation becomes infinite. By changing DC and RF in time, usually at a fixed ratio, ions with different
m/z values can be transmitted to the detector one after another.

Ion trap
An ion trap is a device that uses an oscillating electric field to store ions. There are several common types of ion
trap: 3D ion trap, linear ion trap, orbitrap and Fourier transform ion cyclotron resonance.

1 Quadrupole ion trap (QIT)

Quadrupole ion trap is also called 3 dimension ion trap. The QIT mass spectrometer uses three electrodes to trap
ions in a small volume. It consists of a cylindrical ring electrode and two end-cap electrodes. A mass spectrum is
obtained by changing the electrode voltages to eject the ions from the trap. The end-cap electrodes contain holes
for the introduction of ions from an external ion source and for the ejection of ions toward an external detector.

2 Linear Ion Trap (LIT)

By creating a potential well for the ions, the linear ion trap can be used as a mass filter or as a trap. The linear ion
trap uses a set of quadrupole rods to confine ions radially by a two-dimensional radio frequency (RF) field. And a
static electrical potential can confine the ions axially. They are confined by application of appropriate RF and DC
voltages with their final position maintained within the center section of the ion trap. The RF voltage is adjusted
and multi-frequency resonance ejection waveforms are applied to the trap to eliminate all but the desired ions in
preparation for subsequent fragmentation and mass analysis.

3 Orbitrap
Orbitrap is the newest addition to the family of high-resolution mass analyzer. In orbitrap, moving ions are trapped
in an electrostatic field. The electrostatic attraction towards the central electrode is compensated by a centrifugal
force that arises from the initial tangential velocity of ions. The electrostatic field which ions experience inside the
orbitrap forces them to move in complex spiral patterns. The axial component of these oscillations is independent
of initial energy, angles and positions, and can be detected as an image current on the two halves of an electrode
encapsulating the orbitrap. A Fourier transform is employed to obtain oscillation frequencies for ions with
different masses, resulting in an accurate reading of their m/z.

4 The Fourier Transform Ion Cyclotron Resonance (FT-ICR)

The Fourier Transform Ion Cyclotron Resonance mass spectrometer consists of three main sections: excitation
plates, trapping plates, detector plats, and they consist a cell.
Ions which are affected by a magnetic field move at a cyclotron frequency. After a radio frequency voltage at the
same frequency of cyclotron frequency is applied, the ions absorb energy and accelerate to a larger orbit radius
than their original path. After excitation, the cyclotron radius of ions still remains the larger state. And as the ions
around approach to the top and bottom plate, the electrons travel from top to bottom. The motion of electrons
between these two plates produces a detectable current. The decay over time of the image current resulting after
applying a short radio-frequency sweep is transformed from the time domain into a frequency domain signal by a
Fourier transform.

IONIZATION

Electron Ionization (EI)

EI is one of the first and popular ionization methods for mass spectrometry. It is appropriate for
organic molecules whose relatively molecule weight is below 600. In this technique, a current
passes a wire filament can produce electrons for ionization. And an electric field accelerates
these electrons to produce a beam of high energy electrons. When a molecule passes this high
energy electrons beam, an electron can be expelled from the molecule to produce an ion.
EI works well for many gas phase molecules, so it always works with gas chromatography
(GC), which can be incorporated for the separation of mixtures of thermally stable and volatile
gases. Although EI works well for many gas phase molecules, it does have some disadvantages.
EI causes extensive fragmentation, therefore the molecular ion is not observed for many compounds.

Chemical Ionization (CI)

Chemical ionization is a lower energy process than EI. In CI, ions are produced
by the collision of the samples with ions of a reagent gas that are present in the
ion source. Inside the ion source, the reagent gas is extremely more than
samples. Reagent gas is first subjected to electron impact to produce reagent gas
ions by the fast electrons from the filament. Sample ions are produced by the
ion-molecule reactions of reagent gas ions and sample molecules. In general,
reagent gas molecules are present in the ratio of about 100:1 with respect to
sample molecules. Some common reagent gases include methane, ammonia,
and isobutene. Positive and negative ions of the sample are formed by reactions
with this plasma. Like EI, CI usually works with gas chromatography. CI has the same general limitations as electron
ionization mass spectrometry (EIMS) on the volatility and thermal stability of the compound being analyzed.

MALDI-TOF MASS SPECTROMETER

MALDI stands for Matrix-Assisted Laser Desorption Ionization. In this ionization method samples are fixed in a
crystalline matrix and are bombarded by a laser. The sample molecules vaporize into the vacuum while being ionized
at the same time without fragmenting or decomposing.

TOF stands for Time of Flight, a mass spectrometry method that separates ions by their mass to charge ratio and
determines that mass to charge ratio by the time it takes for the ions to reach a detector.
Working Principle of MALDI-TOF Mass Spectrometry

The MALDI TOF process is a two-phase procedure;

Ionization Phase
Time of Flight Phase
Ionization Phase:
Initially, the samples are fixed in a crystalline matrix in a target plate and are bombarded by a laser. The
sample molecules vaporize into the vacuum while being ionized at the same time. High voltage is then applied to
accelerate the charged particles.
Time-of-flight mass spectrometry phase:

In the linear mode, particles will impinge upon the linear detector within a few nanoseconds after ionization.
Higher mass molecules will arrive later than lighter ones. Flight time measurement make it possible to determine
molecule masses directly. Each peak in the spectrum corresponds to the specific mass of the particle along the time
axis, starting with the ionization moment.
In the reflector mode, the particles are diverted so that they fly towards a second detector. In addition to
extending the flight distance, the reflector also focuses the masses. The combination of these two effects makes for
higher resolution than in the linear mode.

The net result is a generation of a mass spectrum which is compared with those of well-characterized
organisms available in the reference library database to identify the isolate.

Procedure
1. Pick a bacterial colony and smear it onto a target plate.
2. Add 1-2 µl of a matrix consisting α-Cyano-4-hydroxycinnamic acid
(CHCA) dissolved in acetonitrile (50%) and 2.5% trifluoroacetic acid on to it and dry it on
the target plate (at room air)
3. Place the target plate into the plating chamber of the mass spectrometer, close it and
perform the analysis.
Advantages of MALDI-TOF Mass Spectrometry over Conventional Technology
• Significantly decreases the turnaround time. Processing time is similar to rapid biochemicals.
• The sample preparation is simple and the sample requirement is minimal. A single colony is sufficient in
order to generate spectra of sufficient quality
• Cost effective-low consumable costs
• Automated, robust, interlaboratory reproducibility
• Broad applicability (all types of bacteria including anaerobes, fungi)
• Adaptable-open system, expandable by user
Limitations
• Identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of
the type strains of specific genera/species/subspecies/strains
• No susceptibility information is provided
• Not useful for direct testing of clinical specimens (except urine)
• Some organisms require repeat analysis and additional processing (extraction)
• The acceptable score cutoffs vary between studies and some closely related organisms are not differentiated.
• Some organisms currently cannot be reliably identified by this method, such as Shigella spp and
Streptococcus pneumoniae.
Applications of MALDI-TOF
• Pathogen Identification
• Cancer typing directly from serum, tissue extracts, and other bodily fluids
• Tissue imaging –Proteins for cancer typing –Small molecules for drug disposition
• Biomarker Identification and Validation –Mass Spec Immunoassay –Peptide quantitation (SISCAPA and
others)
• Clinical assays of biomarkers for diagnosis and treatment
• QC of synthetic olignucleotides, peptides, proteins and small molecules.
Biochemistry:
MALDI – TOF MS is used in the field of biochemistry to identify the proteins and characterize it because protein
contains a molecular weight that has an intact structure that becomes fragmented when ionized.
Peptide mass fingerprinting (PMF):
Peptide mass fingerprinting is used in the identification of proteins from simple mixture where MALDI – TOF plays
an important role by its simple operation with high resolution and good mass accuracy. Peptides are generated by
enzyme-like trypsin and are analyzed by MALDI – TOF MS and the obtained peptide masses are compared with
the database.
Clinical and Environmental Bacteriology:
For a rapid, reliable, and cost-effective diagnosis of clinical and environmental samples, MALDI – TOF MS is
appropriate for the early identification of bacterial culture. MALDI – TOF can detect blood culture, stool and urine
samples, cerebrospinal fluids, and respiratory tract infections.
It can also identify food and waterborne pathogens and environmental samples.
Detection of viruses:
Besides immunological methods, researchers have proved using MALDI – TOF MS as more advanced and effective
in use for diagnosing infectious viruses like influenza viruses, herpes viruses, and hepatitis viruses.
 First, the viral genetic materials are amplified by PCR and later identified by MALDI – TOF MS which gives rapid
and reliable results in a short period.
Organic chemistry:
Some synthetic macromolecules with a high molecular weight such as catenanes, rotaxanes, and hyperbranched
polymers can be analyzed rapidly with effective results.

PEPTIDE MASS FINGERPRINTING (PMF)

Peptide mass fingerprinting (PMF), also known as protein fingerprinting, is a high-throughput analytical
method that developed in 1933 to identify proteins. Endoproteases first cleaves the unknown target protein into
smaller peptides. The absolute mass of these peptides can be accurately measured using a mass spectrometer (such
as MALDI-TOF), and a list of peptide peaks of the unknown protein is also obtained. This list of peaks is compared
to a list of theoretical peptide peaks from a protein database using a computer program. This program translates all
organism's known genomes into proteins, theoretically digests the proteins into small peptides, and calculates the
absolute mass of the peptide from each protein. At the same time, the mass of the peptide from the lysed unknown
protein is also compared with the theoretical peptide mass of each protein encoded in the genome. The results are
statistically analyzed to find the best match.
The Process of Peptide Mass Fingerprinting

After the proteins in biological tissues or cells are purified, they are separated by 2D or SDS-PAGE gel and
compared using imaging techniques. Proteins with significant differences are selected for further analysis. A series
of peptide compounds that reflect the characteristics of the protein are obtained by degumming and enzymatic
hydrolysis of the selected protein. The fingerprint of the peptide mixture is accurately measured by a mass
spectrometer to obtain the molecular weight of the protein. Searching through a known database provides quasi-
determinism in the selected protein.
The Applications of Peptide Mass Fingerprinting
With the development of analytical chemistry technology, PMF has been widely used in various fields such
as drug authenticity identification, food quality control and human disease diagnosis due to its high sensitivity, high
stability and low sample loss. The World Health Organization and the State Food and Drug Administration of China
use PMF as a material identification and process control technology to provide objective indicators for quality
evaluation.
Advantages and Disadvantages of Peptide Mass Fingerprinting
➢ Advantages
• Does not rely on protein sequencing for protein identification, but the peptide quality.
• Simple and fast, a classic method for the identification of proteins by first-order mass spectrometry
 ➢ Disadvantages
• The protein sequence of interest must exist in the target database.
• Good for single protein analysis. Presence of mixed proteins greatly increases the complexity of the analysis.
• Peptides of similar quality will greatly increase the difficulty of matching.

NUCLEAR MAGNETIC RESONANCE (NMR)

Nuclear Magnetic Resonance Spectroscopy, or “NMR,” is a process used to find out information about a
compound’s magnetic properties. It does so by recording the magnetic spectral patterns given off by the nuclei
within a sample’s atoms. Using NMR, researchers can determine the molecular structure of a compound.
Nuclear magnetic resonance (NMR) spectroscopy is the study of molecules by recording the interaction of
radiofrequency (Rf) electromagnetic radiations with the nuclei of molecules placed in a strong magnetic field.
Swiss scientist Richard Robert Ernst was awarded the 1991 Nobel Prize in chemistry for contributions to the
development of the method of high-resolution nuclear magnetic resonance (NMR) spectroscopy.

Principle
The principle behind NMR is that many nuclei have spin and all nuclei are electrically charged. If an external
magnetic field is applied, an energy transfer is possible between the base energy to a higher energy level (generally
a single energy gap). The energy transfer takes place at a wavelength that corresponds to radio frequencies and when
the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is
measured in many ways and processed in order to yield an NMR spectrum for the nucleus concerned.

Applications of NMR spectroscopy in structural biology - protein structure determination - ligand screening for
drug discovery - analysis of mobility in proteins and protein-ligand interactions - macromolecular complexes -
protein folding – imaging
 NMR is used in biology to study the biofuids, cells, perfused organs and bio macromolecules such as nucleic
acids (DNA. RNA) carbohydrates proteins and peptides, And also labeling studies is biochemistry. NMR is used
physics and physical chemistry to study high pressure diffusion, Liquid crystals liquid crystal solutions, membranes,
rigid solids. NMR is used in pharmaceutical science to study pharmaceutical and drug metabolism.
NMR is a powerful technique applied in a variety of field, especially in biomedical research. NMR can identify
molecules, to probe molecular dynamics and study the molecular interactions. The size and the motion of the
molecules also affect NMR relaxation properties, which are fully exploited in NMR research too.

ELECTRON SPIN RESONANCE (ESR)

Electron spin resonance (ESR) is a spectroscopic technique that is used to detect the transitions induced by
electromagnetic radiation between the different energy levels of electron spins in the рresenсe оf а stаtiс mаgnetiс
field.

• Also called EPR Spectroscopy or Electron Paramagnetic Resonance Spectroscopy

• Non-destructive technique
• Extensively used in transition metal complexes
• Deviated geometries in crystals

Principle
With an ESR instrument, a static magnetic field and microwaves are used to observe the behavior of the
unpaired electrons in the material being studied. The study of the behavior of the electrons in a sample gives
information about the condition of the sample.

ESR is used to observe and measure the absorption of microwave energy by unpaired electrons in a magnetic field.
The phenomenon of electron spin resonance (ESR) is based on the fact that an electron is a charged particle.
It spins around its axis and this causes it to act like a tiny bar magnet. When a molecule or compound with an
unpaired electron is placed in a strong magnetic field the spin of the unpaired electron can align in two different
ways creating two spin states ms = ± ½.
The alignment can either be along the direction (parallel) to the magnetic field which corresponds to the lower
energy state ms = – ½ Opposite (antiparallel) to the direction of the applied magnetic field ms = + ½
The two alignments have different energies and this difference in energy lifts the degeneracy of the electron spin
states. The energy difference is given by:
∆ E = E+ – E- = hv = gmßB
Where,
h = Planck’s constant (6.626 x 10-34 J s-1)
v = the frequency of radiation
ß = Bohr magneton (9.274 x 10-24 J T-1) B = strength of the magnetic field in Tesla
g = the g-factor which is a unit less measurement of the intrinsic magnetic moment of the electron, and its value for
a free electron is 2.0023.
Applications of Electron Spin Resonance (ESR)
Electron spin resonance (ESR) spectroscopy has been widely applied in the research of biological free radicals
for quantitative and qualitative analyses of reactive oxygen species (ROS) and reactive nitrogen species (RNS). This
method is now widely used as one of the most powerful tools for free radical studies.
• ESR spectrometry is one of the main methods to study transition metal containing metalloproteins.
• To determine the rate of catalysis
• To know about the active site geometry
• To study denaturation and protein folding
• In studies relating to enzyme-ligand interaction
• In Biological Systems
• Study of Free Radicals
• Spin Labels
• Study of Inorganic Compounds
• Reaction Velocities & Mechanisms
• Study of naturally occurring substances such as minerals with transition elements, minerals with defects (e.g;
quartz), Hemoglobin (Fe), Petroleum, Coal, Rubber etc.
• Conducting Electrons
INFRARED (IR) SPECTROSCOPY
Infrared spectroscopy (IR) relies on the fact that most molecules absorb light in the infrared region of the
electromagnetic spectrum, converting it to molecular vibration. This absorption is characteristic of the nature of the
chemical bonds present in a sample.
With a spectrometer, this absorption is measured as a function of wavelength (as wave numbers, typically from 4000
- 600 cm-1). The result is an IR spectrum that serves as a characteristic "molecular fingerprint" whichcan be used to
identify organic and inorganic samples.

Principle of Infrared Spectroscopy

The IR spectroscopy theory utilizes the concept that molecules tend to absorb specific frequencies of light
that are characteristic of the corresponding structure of the molecules. The energies are reliant on the shape of the
molecular surfaces, the associated vibronic coupling, and the mass corresponding to the atoms.
For instance, the molecule can absorb the energy contained in the incident light and the result is a faster
rotation or a more pronounced vibration.

Applications of Infrared (IR) Spectroscopy

Infrared spectroscopy has proved to be a powerful tool for the study of biological molecules, including proteins,
lipids, carbohydrates, and nucleic acids. ... Biological systems, including animal and plant tissues, microbial cells,
clinical samples, and food, have all been successfully studied using infrared spectroscopy.
• It has been of great significance to scientific researchers in many fields such as:
• Protein characterization
• Nanoscale semiconductor analysis and
• Space exploration.
• Analysis of gaseous, liquid or solid samples
• Identification of compounds
• Quantitative analysis
• Information regarding functional groups of molecules and constitution of molecules can be deduced from IR
spectrum
• To know about interaction among molecules