1.
01 Molecular Techniques In Protein Analysis
DV NILLASCA || December 2023
Introduction
• A protein must be purified before its structure and the mechanism of
its action can be studied. A daunting problem in this case is the
variation in size, charge and water solubility of proteins.
• No single method can be used to isolate all proteins. Different
techniques are employed in separating proteins and detecting their
presence.
PROTEIN ANALYSIS
WESTERN BLOTTING
• Modifications of the Southern blot in which the immobilized targets are
proteins. SDS-PAGE (Sodium dodecyl-sulfate polyacrylamide gel
electrophoresis)
• Detects proteins and very small DNA fragments; SDS eliminates the
influence of protein large structure and charge.
• Addition of SDS to increase resolution and eliminate larger proteins.
• Use of polyacrylamide gel – used to separate nucleic acid fragments
smaller than 10 bp.
ELISA (Enzyme – linked immunosorbent assay)
• Immunological assay used to screen and measure Ab, Ag, proteins &
GP
• Proteins and GP will serve as epitopes for antibodies added to
reaction.
• Proteins have the highest antigenicity as compared to other
substances.
MALDI-TOF (Matrix-assisted laser desorption/ionization-
time of flight)
• Detect bacteria and proteins using either intact or cell extracts
• Aids in the classification of bacteria and their taxonomy
WESTERN BLOTS \
• Detects proteins in serum, cell lysate, or extract are separated on:
• SDS-polyacrylamide gels (SDS-PAGE) – resolves proteins according
to molecular weight
• Isoelectric focusing gels (IEF) - differentiates them according to
charge
Cell lysate - the extracted DNA through cell lysis
Electrophoresis - method for resolution of different fragments: SDS
PAGE or IEF
Western Blots
• The main application of Western blot method is to confirm enzyme
linked immunoassay results for human immunodeficiency virus (HIV)
and hepatitis C virus among other organisms.
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Molecular Biology
• In this procedure, known HIV proteins are separated by
electrophoresis and transferred and bound to a nitrocellulose
membrane.
• The patient’s serum is overlaid on the membrane, and antibodies with
specificity to HIV proteins bind to their corresponding protein.
• Unbound patient antibodies are washed off, and binding of antibodies
is detected by adding a labelled anti human immunoglobulin antibody.
• If HIV antibodies are present in the patient’s serum, they can be
detected with anti- human antibody probes appearing as a dark band
on the blot corresponding to the specific HIV protein to which the
antibody is specific.
SDS-PAGE:
1. Routine testing requires diluting the sample with denaturants (1:1)
like 0.04 M Tris HCl, pH 6.8, 0.1% SDS.
2. Depending on the complexity of the protein, polyacrylamide
concentrations vary 5%-20%.
• 5-20% Polyacrylamide gel: electrophoretic medium
3. 1–50 μg of protein is loaded per well. Before loading, the sample is
treated with denaturant.
• Protein concentration varies
4. Pre-stained molecular weight standards are run with the samples
• To orient the membrane after transfer and to approximate the sizes of
the proteins after probing.
• Standards used would vary ranging from 11,700 to 205,000 daltons.
5. Pre-treatment of gel with mild buffers such as 20% glycerol in
• 50 mM Tris-HCl, pH 7.4, to renature proteins before transfer.
• The gel system used may affect subsequent probing of proteins with
antibodies. Denaturing gels could affect antigenic sites on the
protein such that they will not bind with the labelled antibodies.
6. After electrophoresis, proteins can be blotted to membranes by
capillary or electrophoretic transfer.
•Nitrocellulose has high affinity for proteins and is easily treated with
detergent to prevent binding of the primary antibody to the membrane
itself before hybridization. (Nitrocellulose - membrane used)
• Aside from capillary or electrophoretic transfer, vacuum
transfer can also be used.
• The accuracy and sensitivity of the separation can be enhanced by
using a combination of IEF gels followed by SDS-PAGE. The standards
used in Western Blot vary according to usage but usually these range
[MOLECULAR BIO] 1.01 Molecular Techniques in Protein – DV NILLASCA
from 11,700 daltons (cytochrome C) to 205,000 daltons (myosin). amplify DNA and RNA. Same goes with the Western blot

Standards are commercially available. technique.

• Protein probes used in this method are antibodies that bind

specifically to the immobilized target protein.

 Polyclonal or monoclonal antibodies can be used.

POLYCLONAL ANTIBODIES

• Are made by immunization with a specific antigen, usually a

peptide or protein.

• The immunoglobulins reduced are subsequently isolated

from serum by affinity chromatography.

• Polyclonal antibodies are a mixture of immunoglobulins that

are directed at more than one epitope on the antigen.

• Protein loaded could vary depending on the sample and the use of the
• In Western blot technology, polyclonal antibodies can give a
analysis.
more robust signal especially if the target epitopes are partially lost
• The standards are important as the basis for the result of the
during electrophoresis and transfer.
separated proteins.
MONOCLONAL ANTIBODIES
• Denaturation could affect the antigen sites in the proteins by
• Are first demonstrated in spleen cells from immunized mice fused with
preventing the attachment of the labeled
mouse myeloma cells to form hybrid cells (hybridomas) that could grow
antibodies.
in culture and secrete antibodies.
• Nitrocellulose and other membranes presented last time in the
• Monoclonal antibodies are then isolated from cell culture fluid by
hybridization techniques could be used. After transfer or blotting, we will
chromatography.
be probing this with the antibody.
• Monoclonal antibodies are more specific and may give less
• There will be immunostaining of the blot in the membrane.
background.
• It will be detected by autoradiography.
• Specific to one target - antigen or epitope of the antigen.
• The autoradiograph will provide a signal or product. There will be
• One drawback of monoclonal antibodies however when the targeted
antigen bands that could be visualized as a product of the Western Blot
epitope is lost, these antibodies have nothing to bind and no signal is
process.
generated.
• Antigen can be run on electrophoresis first, either isoelectrofocusing
• The sample should be handled carefully.
or SDS-PAGE. Or it can be done sequentially.

• This is to resolve the proteins. The standard/marker on the left side is

needed to know the protein being identified.

• Then, these bands on the gel would be transferred to a membrane

through blotting. Usually the membrane is nitrocellulose.

• Capillary Transfer is the type of blotting technique in the picture,

movement is upward from the gel to the paper.

• After transfer to the membrane, we would then perform

immunostaining of the blot. Proteins in the membrane will serve as the

antigen. The labelled antibody from the reagent is added to the

• Primary antibody (probe) will be added to the target protein. Then, the
membrane. This would attach if the specific protein is present.
secondary antibody bound to a label will be added. This is important for
• The labelled antibodies can be detected by autoradiography.
detection.
PROTEIN PROBES • The secondary antibody is called the anti-antibody.
We have discussed in Topic 3 that we need probes to

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[MOLECULAR BIO] 1.01 Molecular Techniques in Protein – DV NILLASCA
• The Fab region of the anti-antibody is attached to the Fc region of the • Agarose is a linear polymer of agarobiose, which consists of 1,3-

primary antibody. This can also happen with just the primary antibody linked--D-Galactopyranose and 1,4- linked 3,6-anhydro-αL-

with a label. galactopyranose.

• Secondary antibody may be absent • The concentration of the agarose dictates the size of the spaces in the

• Polyclonal Antibody is more preferred than the monoclonal gel

• During the transfer and blotting, proteins may be washed off. Since • Agarose concentrations above 5% and below 0.5% are not practical.

the monoclonal antibody is specific to a single epitope, once it loses the • High-concentration agarose will impede migration

target protein, it no longer has a purpose. • Very low concentrations produce a weak gel with limited integrity. and

• Proteins contain many epitopes. will, therefore, be determined by the size of DNA and proteins.

• Secondary antibody may be absent

• Polyclonal Antibody is more preferred than the monoclonal • During

the transfer and blotting, proteins may be washed off. Since the

monoclonal antibody is specific to a single epitope, once it loses the

target protein, it no longer has a purpose.

• Proteins contain many epitopes.

PULSE FIELD GEL ELECTROPHORESIS
• We have encountered the term electrophoresis a lot and you have
• For genomic-sized DNA molecules (very large DNA with 50,000–
tackled this in your Clinical Chemistry lecture. Electrophoresis is the
250,000 bp which cannot be resolved efficiently by simple agarose
movement of molecules by an electric current.
electrophoresis).
• It is practically done in a matrix to limit migration and contain the
• Pulses of current applied to the gel in alternating dimensions enhance
migrating material. Electrophoresis is routinely applied to the analysis of
migration.
proteins and nucleic acids.
• This can be incorporated in the agarose gel.
• Gel Systems
• The simplest approach to this method is field inversion gel
• Buffer Systems
electrophoresis (FIGE).
• Electrophoresis Machinery
• Works by alternating the positive and negative electrodes during
• Gel Loading
electrophoresis
• Detection Systems
• DNA goes periodically forward and backward.
• Sources of Error
POLYACRYLAMIDE GEL (PAGE)
GEL SYSTEM • Another matrix for electrophoresis best designed for proteins and very
• Gel matrices provide resistance to the movement of molecules under
small DNA fragments.
the force of the electric current.
• Acrylamide, in combination with the cross-linker methylene
• They prevent diffusion and reduce convection currents so that the
bisacrylamide, polymerizes into a gel that has consistent resolution
separated molecules form a defined group, or “band.”
characteristics.
• The gel can then serve as a support medium for analysis of the
• Polyacrylamide was originally used mostly for protein separation, but it
separated components.
is now routinely applied to nucleic acid analysis.
• Characteristics of a good gel matrix include being unaffected by
• Polyacrylamide gels can also be used for sequencing nucleic acids,
electrophoresis, simple to prepare and amenable to modification.
mutation analyses, nuclease protection assays, and other applications
Agarose and polyacrylamide are polymers that meet these criteria.
requiring the resolution of nucleic acids down to the single-base level.

• Acrylamide is supplied to the laboratory in several forms:

AGAROSE GEL
• Agarose is a polysaccharide polymer extracted from seaweed.
• It is a component of agar used in bacterial culture dishes.
1. The powdered form is a dangerous neurotoxin and must be handled
with care.
2. Solutions of mixtures of acrylamide and bisacrylamide are less

hazardous and more convenient to use.

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[MOLECULAR BIO] 1.01 Molecular Techniques in Protein – DV NILLASCA
• Polymerization of gel SDS (PAGE)
• One modification of this matrix is the which entails usage of sodium
• Unlike agarose gels that polymerize upon cooling, polymerization of
dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and
polyacrylamide gels requires the use of a catalyst.
polyacrylamide
• Polymerization of gel
• Eliminate the influence of the protein's large structure and charge.
• Unlike agarose gels that polymerize upon cooling, polymerization of
• This system allows proteins to be separated solely based
polyacrylamide gels requires the use of a catalyst.
on polypeptide chain length.
• The catalyst may be the nucleation agents, ammonium persulfate

(APS) plus N,N,N,N-tetramethylethylenediamine (TEMED), or light

activation.

• APS produces free oxygen radicals in the presence of TEMED to

drive the free-radical polymerization mechanism.

• Free radicals can also be generated by a photochemical process

using riboflavin plus TEMED. SDS POLYACRYLAMIDE POLYACRYLAMIDE

• Excess oxygen inhibits the polymerization process. • is a detergent with a strong • Polyacrylamide forms a mesh-

• Therefore removal of air of the gel solution is often done before the protein denaturing effect and like matrix suitable for the

addition of the nucleation agents. binds to the protein backbone at separation of proteins of typical

• Polyacrylamide gels for nucleic acid separation are very thin making a constant molar ratio. size. The strength of the gel

gel preparation difficult. • In the presence of SDS and a allows easy handling. When

• Systems have been designed to facilitate the preparation reducing agent that cleaves proteins are separated, smaller

of single and multiple gels. disulfide bonds critical for proper proteins migrate faster due to

• Increasing numbers of laboratories are using pre-formed folding, proteins unfold into less resistance from the gel

polyacrylamide gels to avoid the hazards of working with acrylamide linear chains with negative matrix.

and the labour time involved in gel preparation. charge proportional to • Other influences on the rate of

• However, use of pre-formed gels must be scheduled since the product the polypeptide chain length. migration through the gel matrix

has a limited shelf life. include the structure and charge

of the proteins

• The main advantage of polyacrylamide over agarose is the higher

resolution capability for small fragments that can be accomplished with

polyacrylamide.

• Another advantage of polyacrylamide is that, unlike agarose, the

components of polyacrylamide gels are synthetic; thus, there is not as

much difference in batches obtained from different sources.

• Further, altering monomers in a polyacrylamide gel can change the

pore size and, therefore, the sieving properties can be modified in a

General Scheme of SDS-PAGE. The reducing agent for this system is
predictable and reproducible manner.
2-mercaptoethanol. Also, see that the number of SDS that attaches to

the protein linear structure is proportional to chain length.

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[MOLECULAR BIO] 1.01 Molecular Techniques in Protein – DV NILLASCA
1. First, we would be disrupting the disulfide bonds between or within •This is accomplished through the electrochemica l characteristics of

the protein molecule to unfold the proteins. the buffer components.

• The disulfide bond will be the reason why the proteins will fold. So •The pH of a buffered solution remains constant as the buffer molecules

upon the breakdown of disulfide bonds, proteins will be unfolding take up or release protons given off or absorbed by other solutes.

resulting in a linear peptide chain.

•Uses 2-mercaptoethanol

2. Add a reducing agent, 2-mercaptoethanol, to reduce or break the

disulfide bonds in the system

3. Add a number of SDS with a negative charge which would attach to

the linear protein structure. This would be proportional to the length of

the polypeptide chain.

• The amount of SDS (blue circles) that would attach to the polypeptide

chain is proportional to the length of the polypeptide chain.

• Molecular weight of protein is identified through the length of the

polypeptide chain.

4. This would then be resolved by electrophoresis. Marker in

electrophoresis is the basis of our results.

• Polyacrylamide gel electrophoresis

• Once the gel polymerizes, the cassette is mounted into an apparatus

so that the top and bottom edges are placed in contact with buffer

chambers containing a cathode and an anode, respectively.

• The blue color would be the buffer solution. The cassette would be

attached to the upper and lower buffer reservoir. The lower buffer will

contain the anode electrode while the upper buffer will contain the

cathode electrode.

• The running buffer contains ions that conduct current through

the gel.

• When proteins are loaded into wells at the top edge and current is

applied, the proteins are drawn by the current through the matrix slab

and separated by the sieving properties of the gel.

See that low molecular weight proteins are faster to migrate compared
• The separating properties are dependent on the sieving properties of
to high molecular ones due to less gel resistance.
the gel.
• Proteins migrate to the Anode
• A stacking gel is cast over the top of the resolving gel – To obtain
• Low MW protein migrates faster than High MW proteins
optimal resolution of proteins
• Agarose gel: natural source from seaweeds and is used as a culture
• The stacking gel has a lower concentration of acrylamide, lower pH,
medium in agar
and a different ionic content compared to separating gel.
• Polyacrylamide gel: synthetic however it’s delicate to be handled
• This allows the proteins in a loaded sample to be concentrated into
since it is very thin. It can resolve proteins properly and small fragments
one tight band during the first few minutes.
of DNA. Fresh preparation is preferred because of its short shelf life.
• Without the stacking gel, upon sample loading it will already start to
BUFFER SYSTEMS
•The purpose of a buffer system is to carry the current and protect the migrate downwards. Stacking gel serves as a boundary.

samples during electrophoresis.

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[MOLECULAR BIO] 1.01 Molecular Techniques in Protein – DV NILLASCA
• Stacking gel - line up all the protein samples, so that they can enter BIS – TRIS SYSTEM

the resolving gel at the same time In the case of the NuPAGE® Bis-Tris system, three ions are primarily

• Resolving gel - separate the proteins based on their molecular involved:

weight. 1. CHLORIDE (-)

SDS- PAGE BUFFER SYSTEM • supplied by the gel buffer

• Utilizes a discontinuous buffer system
• serves as the fast-moving leading ion.
• To concentrate samples into a very sharp zone in the stacking gel at
2. MES or MOPS (-) (depending on the running buffer choice)
the beginning of the run.
• serves as the trailing ion
• In a discontinuous buffer system, the primary anion in the gel is
• MES: 2-(N-morpholino) ethane sulfonic acid
different (or discontinuous) from the primary anion in the running buffer.
• MOPS: 3-(N-morpholino) propane sulfonic acid
• Both the NuPAGE® systems (Bis-Tris and Tris-Acetate gels) and the
3. BIS-TRIS (+)
Laemmli (Tris-Glycine) system are examples of discontinuous buffer
• acts as the common ion present in the (only on) gel
systems and work in a similar fashion. However, the NuPAGE® system
• The combination of a lower pH gel buffer (pH 6.4) and
operates at a lower pH as a result of different ions in the system.
running buffer (pH 7.3 - 7.7) leads to a significantly lower
TRIS-GLYCINE SYSTEM operating pH (pH 7.0) during electrophoresis.
Three ions are primarily involved:

1. CHLORIDE (-)

• supplied by the gel buffer

• serves as the leading ion

• has the highest attraction to the anode relative to other anions in the

system.

2. GLYCINE (-)

• the primary anion provided by the running buffer

• serves as the trailing ion

• only partially negatively charged and remains behind the more highly

charged chloride ions in a charged environment.

3. TRIS BASE (+) TRIS – ACETATE SYSTEM

• In the case of the NuPAGE® Tris-Acetate system, three ions are
• is a common ion present in both the gel and the running buffers.
primarily involved:
• During electrophoresis, the gel and buffer ions in the Tris-Glycine
• Acetate (-), the leading ion from the gel buffer
system form an operating pH of 9.5 in the separating region of the gel.
• Tricine (-), the trailing ion from the running buffer

• Tris (+), the common ion (in both gel and running buffer)

• This system also operates at a significantly lower pH than the Tris-

Glycine system at pH 8.1.

Tris-Glycine Operating System Tris-Acetate Operating System

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SDS-PAGE BUFFER SYSTEM • One effective way of “unstacking” is to use a shift in pH to accelerate
As you can see, the discontinuous buffer systems
the trailing ions.
• Employ different buffer ions and pH in the gel and in the electrode
• For the glycine/chloride system, a stacking gel is cast so that it will
reservoirs (buffer). Samples are loaded first onto the stacking gel, which
regulate to a lower pH than the resolving gel. When the glycine enters
overlays a smaller pore resolving gel.
the higher pH resolving gel, its net negative charge will increase its
• The major advantages of discontinuous buffer systems are that
effective mobility.
relatively large volumes of dilute protein samples can be applied to the
• Consequently, the glycine overtakes some of the proteins and now
gel and resolution is much greater.
migrates directly behind the chloride ions, causing proteins to resolve
• This increased resolution is a direct result of the way proteins
according to their size.
concentrate, or stack, into narrow zones during migration through the

large-pore stacking gel, and de-stack or resolve, under the given

conditions in the small pore resolving gel.

• Stacking gel is important in order for the bands to be concentrated

before migration. This would result in a better resolution of the different

migration of the bands that would represent the proteins.

• At a given pH, only part of a population of protein molecules will be

dissociated at any time.

ELECTROPHORESIS MACHINERY
• The velocity at which these molecules migrate will be dependent upon • Gel electrophoresis can be done in one of two conformations,

the effective mobility and the voltage gradient. horizontal or vertical. In general, agarose gels are run horizontally, and
• Therefore, an ion of lower mobility can migrate as fast as one with polyacrylamide gels are run vertically.
higher mobility if the products of voltage and effective mobility are equal.

• For example, in the Ornstein and Davis system, the sample and

stacking gel contain Tris-HCl buffer whereas the upper buffer reservoir

contains Tris-Glycine buffer.

• At the pH of the sample buffer and stacking gel (pH 6.7), glycine is

weakly ionized and therefore its mobility is low. Chloride is completely

ionized and has a much higher mobility, while the mobility of proteins is

intermediate between the two.

•Horizontal gels are run in acrylic containers called gel boxes or baths
• Once a voltage is applied, chloride (leading) ions migrate away from
that are divided into two parts with a platform in the middle on which the
the glycine (trailing) ions leaving behind a zone of lower conductivity, a
gel rests.
higher voltage gradient, and higher pH.
•Platinum wires make up the electrodes in the gel compartments.
• The zone accelerates the glycine so it keeps up with the chloride ions.
• The wires are connected to a power source by banana clips or
As this glycine/chloride boundary moves through the sample and
connectors through the walls of the container.
stacking gel, any proteins in front are rapidly overtaken by chloride ions
• The gel in the box is submerged with an electrophoresis buffer filling
which have a higher velocity.
both compartments and making a continuous system through which the
• Behind the moving boundary, the proteins have a higher velocity than
current flows.
glycine.
• The thickness of the gel and the volume of the buffer affect migration,
• Therefore, the moving boundary sweeps up the proteins,
so these parameters should be kept constant for consistent results.
concentrating them into stacks in order of decreasing mobility
• As the gel is submerged through the loading and electrophoresis
• As proteins migrate into the resolving gel, they are separated
process, horizontal gels are sometimes referred to as submarine gels.
according to size. But what happens next since all the proteins have
• The power supply will deliver voltage, setting up a current that will run
been stacked at the stacking gel?
through the gel buffer and the gel, carrying the charged sample through
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the matrix of the gel at a certain speed. These gels are shaped as

square or rectangular slabs of varying size. Purchased gel boxes come

with casting trays that mold the gel to the appropriate size for the gel

box.

GEL LOADING
• Long, narrow gel-loading pipette tips are used to deposit the sample

neatly on the floor of the well and these increase band resolution and

sample recovery.

• Vertical gels are casted between glass plates that are separated by

spacers.
• Vertical gel boxes have separate chambers that are connected by the
• The spacers determine the thickness of the gel, ranging 0.05–4 mm.
gel itself.
• The bottom of the gel is secured by tape or by a gasket in specially
• Electrodes are attached to the upper and lower buffer chambers to set
designed gel casting trays.
up the current that will run through the gel.
• After addition of polymerization agents, the liquid acrylamide is poured
• The gel must be in place before filling the upper chamber with a buffer.
between the glass plates with a pipet or a syringe into several wells
• Some systems have a metal plate attached to the back of the gel to
(usually 10 in number with capacity of 50 μL).
maintain constant temperature across the gel. This is called “gel
• The comb is then placed on the top of the gel.
smiling” because similar-sized bands in the cooler outer lanes will
• During this process, it is important not to introduce air into the gel or
migrate slower than comparable bands in the inside lanes.
beneath the comb.
 Prevent gel smiling through maintenance of a constant
• Bubbles:
temperature across the gel.
1. will form discontinuities in the gel
• That’s why one should ensure that there is no variation in temperature
2. oxygen inhibit the polymerization of the acrylamide
across the gel to prevent the undesirable gel smiling from occurring.
• The comb is of a thickness equal to that of the spacers so that the gel
• Vertical gel systems can range from large sequencing systems (35cm
will be the same thickness throughout. After that, these combs are
X 26cm) to mini-systems (8cm X 10cm).
removed.
• Mini-systems are used extensively for analyses that do not require

single base pair resolution. The larger systems are used for sequencing COMBS
A. SHARK’S-TOOTH COMBS - Specialized combs
or other procedures requiring single-base resolution.
• are often used for sequencing gels
 Buffer system is found on both the upper and lower part
• These combs are placed upside down (teeth up, not in contact with

the gel) to form a trough on the gel during polymerization.

• After polymerization is complete, the comb is removed and placed

tooth-side down on top of the gel for loading. With this configuration, the

spaces between the comb teeth form the wells as opposed to the teeth

themselves forming the wells in the horizontal gels.

• Standard combs: used in electrophoretic processes

• Shark’s tooth: used for DNA sequencing

ELECTROPHORESIS MACHINERY

❑ Happens when similar-sized bands in the cooler outer lanes will

migrate slower than comparable bands in the inside lanes.

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A. “MARKER” OR STANDARD LANE
• label the molecular sizes of the bands to avoid confusion.

• Commercially available markers come with a picture of the band

pattern to expect along with the molecular weights of each band.

• Bands are the dark horizontal bars which are actually stained protein

embedded in the gel.

• There would be cases where the band in the sample is not totally in-

B. STANDARD COMBS sync with that of the standards. For example, a band in lane 4 that

• Are removed before the gel is loaded, whereas the shark’s- tooth resides just below the line extended from the 25- kilodalton marker

combs are made so that the wells can be loaded while the comb is in band would suggest that the lane 4 band is almost but not quite 25

place. kilodaltons in molecular weight.

• When the standard combs are removed from the gel, care must be B. PROTEINS’ LANE
• Make a list of proteins for each lane. Then, list the estimated
taken not to break or displace the “ears” that were formed by the
molecular weight of each band in the lane.
spaces between the teeth in the comb that separate the gel wells.
• Lanes with one band indicate that the sample contains only one
Combs
• Polyacrylamide gels can also be cast in tubes for isoelectric focusing protein.

or two-dimensional gel electrophoresis. The tubes containing the gels • Lanes with multiple bands indicate the presence of multiple proteins.

are placed into a chamber separated as for vertical slab gels. This gel Bands that run with the migration front are smaller than suggested by

configuration, however, can run only one sample per gel. the nearest marker and likely cannot be predicted.

• Avoid formation of bubbles so that the area of the wells is equal. If it is • In the proteins list, note oddities. A "smeared“ appearance can

not equal, the sample would have more concentration than the other. It indicate that too many proteins are present or that the viscosity of the

is used in polyacrylamide gel because the placement is vertical. sample affected its migration - Remedy: dilute the sample

• If bands seem to go beyond the edge of the lane or are quite large
DETECTION SYSTEM
• For the procedures and principles discussed above, protein molecular compared with other bands, then the concentration of that protein is

weight alone determines the migration speed of proteins as they move likely too high and should be diluted in future electrophoresis.

through the gel toward the positively charged pole. • A grayish tint throughout the lane, darker than the background gel

• Multiple proteins in the same sample will, therefore, separate from color, indicates indistinguishable protein fragments.

each other and migrate to different positions. • Contaminants are present

• Multiple bands would represent different proteins while a single • Determine the identity of the proteins in each lane. Although this is

protein would represent only one protein. done using only molecular weight, the source of each lane will likely

• On the other hand, presence of one band in the sample indicate clues as well.

electrophoresis gel would indicate that only one protein is present. • Consider that under some conditions, proteins can maintain a dimer or

• "Top" is the location of the wells where the samples were originally trimer association on a gel.

added. • Therefore, one protein may appear on a gel as three distinct bands.

• "Bottom" is where the samples migrated toward and most often The relative darkness of the bands can imply the concentrations of the

contains the dye front that indicates the migrating front of the samples. proteins in solution. Any unknown proteins can be isolated directly from

• Either the left or the right should contain a "marker" or standard, which the original gel and sent for identification.

is used as a predictable molecular weight guide. SOURCES OF ERROR IN ELECTROPHORESIS

• Label the samples for each lane. Across the top, the samples added 1. SAMPLE CONTAMINATION

to the wells will have migrated vertically in "lanes.“ Therefore, all the • If there is foreign DNA or protein in the sample, the gel will have more

bars visible in a vertical column came from the one sample loaded bands than would be found in a gel that contains only the purified

directly above it. sample.

• This is most likely a sample mishandling.

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2. ANOMALIES WITH THE GEL, CURRENT OR BUFFER • If the antigen is in the solid phase, we are detecting the antibody that

• If the concentration of gel is too high or too low, the fragments will is present in the serum.

migrate either too slowly or too quickly. • The antibody will bind specifically to the solid phase present in the

• The voltage must always be steady when running to avoid any well.

fluctuations that will result in unsteady migration of DNA or protein • The primary antibody comes from the patient sample.

fragments. • After a wash step, an enzyme-labelled antiglobulin is added. This

• The buffer solution must also be of the correct composition, as a second antibody reacts with any patient antibody that is bound to the

buffer with the wrong pH or ionic concentration will change the shape of solid phase.

the DNA fragments and proteins. • Washing step: to remove unbound antibodies

3.IMPROPER VISUALIZATION • Second antibody: serves as the anti-antibody, does not bind to the

• If the concentration of the dye or radioactive probe used to visualize solid phase

the samples is too high, the resulting image will be very messy, as • The Fab region of the secondary antibody binds with the Fc region of

residual fragments will also be visualized. the primary antibody.

• If it is too low, there will be no visualization. • If no patient antibody is bound to the solid phase, the second labelled

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) antibody will not be bound and will remain floating in the sample.
• Another method for detection of proteins in bodily fluids
• After a second wash step, the enzyme substrate is added.
• Considered a noncompetitive because the enzyme-labelled reagent
• The amount of enzyme label detected is directly proportional to the
does not participate in the initial antigen–antibody binding reaction.
amount of antibody in the specimen.
• This immunological assay is commonly used to screen and measure
• If the AB is present in the sample, it would bind to the antigen in the
antibodies, antigens, proteins and glycoprotein.
microtiter well. Upon addition of enzyme-labelled immunoglobulin, it
• Ab and Ag are used
would bind to each other giving off an amount of signal to be formed
• Some applications include diagnosis of HIV infection, pregnancy tests,
and detected.
and measurement of cytokines or soluble receptors in cell supernatant
• If it is specific, it would give a signal (either fluorescence or
or serum.
chemiluminescence).
• It remains to be one of the most frequently used immunoassays in the
• This type of assay has been used to measure antibody production to
clinical laboratory due to its sensitivity, specificity, simplicity, and low
infectious agents that are difficult to isolate in the laboratory and has
cost.
been used for autoantibody testing.

• Viral infections especially are more easily diagnosed by this method

than by other types of testing.

• Used as a confirmatory test in the screening of viral infection present

in samples in blood bags during blood donation.

• This technique remains the preferred screening method for detecting

antibodies to HIV, hepatitis A, hepatitis C and Epstein-Barr–specific

antibodies produced in infectious mononucleosis.

General Scheme of ELISA

SO HOW DOES THIS ASSAY TAKE PLACE?

• Either antigen or antibody may be bound to solid phase (either

microtiter plates, nitrocellulose membranes or magnetic latex beads).

• Most of the ELISA tests use antigen bound to solid phase. Patient

serum with unknown antibody is added and given time to react.

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[MOLECULAR BIO] 1.01 Molecular Techniques in Protein – DV NILLASCA
• The target here is the patient’s antigen. • Matrix assisted - samples will be mixed with the matrix and attached

• Sandwich ImmunoAssay - antibody-antigen-antibody to the matrix and the two will be crystalized in a well/plate.

• Solid support is used where the specific capture antibody is attached. • Desorption - matrix and sample crystals are bombarded/hit by the

• The specific capture antibody will capture the antigen if it is present in laser beam.

the patient’s sample. • sample attached from the crystalline matrix will be released

• If it forms an immune complex, an enzyme labeled detection antibody • After the release, the samples become ionized/protonated, and

(secondary antibody) will be added. become positively charged by the laser beam.

• Upon the addition of the substrate, it will give off a colored reaction. • Ionized samples/ analytes are then separated based on the Mass to

• The samples frequently used but not limited to are urine, serum or cell charge ratio.

supernatant. • TOF is used for detection of mass to charge ratio • used in detection

• The second antibody/detection antibody is always labelled with an of bacteria.

enzyme, usually horseradish peroxidase or alkaline phosphatase. • Final Product measured by Mass Spectrometry.

EXAMPLES OF SUBSTRATES: • TOF is used If bacterial protein is detected

• The highlight in each substrate is the colored product present if the
HOW DOES THIS TECHNIQUE WORK?
target antigen is present in the sample
The sample for analysis by MALDI-MS is prepared by mixing with a
1. PNPP (p-Nitrophenyl Phosphate, Disodium Salt)
solution of an energy absorbent, organic compound called matrix.
2. ABTS (Azinobis ethylbenzothiazoline-6-sulfonic acid-diammonium
1. The matrix crystallizes on drying and that leads also to the
salt)
sample entrapped within to co-crystallize.
3. OPD (o-phenylenediamine dihydrochloride) 4. TMB
• Sample is entrapped in the crystallize matrix
(tetramethylbenzidine)
2. A laser beam will then be used to ionize the matrix in an
5. ONGP (ortho-Nitrophenyl-β-galactoside).
automated mode.
• Determination of antigen concentration in a sample requires
• Sample is inside if the matrix is ionized
production of a standard curve using antigens of a known concentration.
3. Desorption and ionization with the laser beam generates
• In preparing for a standard curve, the antigen or the substances
singly protonated ions from analytes in the sample.
should have a known concentration so that it can be easily be plotted.
• Analytes will become protonated ions inside the sample
• The concentration of antigen in a sample can then be calculated using
4. The protonated ions are then accelerated at a fixed potential,
the optical density (OD)
where these separate from each other on the basis of their
MALDI - TOF mass-to-charge ratio (m/z).
• In today’s setting, microorganisms are best identified using 16S rRNA
• Protonated ions on the sample would separate from each
and 18S rRNA gene sequencing techniques.
other based on the mass to charge ratio.
• However, a potential tool for microbial identification and diagnosis was
5. The charged analytes are then detected and measured using
developed in recent years.
different types of mass analyzers like quadrupole mass
• Matrix Assisted Laser Desorption Ionization-Time of Flight Mass
analyzers, ion trap analyzers or time of flight (TOF) analyzers.
Spectrometry (MALDI-TOF MS) had emerged to detect bacteria and
• For microbiological applications mainly TOF mass analyzers
proteins as well.
are used.
• During the MALDI-TOF MS process, microbes are identified
• Used to detect microorganisms.
using either intact cells or cell extracts.
MALDI-TOF ANALYSIS
• The process is rapid, sensitive, and economical in terms of both
•The m/z ratio of an ion is measured by determining the time required
labour and costs.
for it to travel the length of the flight tube.
• Unique application - identifies microbes either intact cells or cell
•A few TOF analyzers incorporate an ion mirror at the rear end of the
extracts.
flight tube, which serves to reflect back ions through the flight tube to a

detector.

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[MOLECULAR BIO] 1.01 Molecular Techniques in Protein – DV NILLASCA
•Thus, the ion mirror not only increases the length of the flight tube, it laboratory, aided by the availability of many commercial libraries of

also corrects small differences in energy among ions. organism PMFs.

•Based on the TOF information, a characteristic spectrum called APPLICATIONS OF MALDI-TOF IN BACTERIOLOGY
1. Clinical Bacteriology (detection of UTI, bacterial meningitis, infectious
peptide mass fingerprint (PMF) is generated for every analyte in the
diarrhea)
sample. PMF will be the basis of TOF for every analyte in the sample
2. Food-and-Water-Borne bacteria
that is protonated ion, TOF is measured, to determine the specific
3. Environmental and Agricultural Bacteriology
analyte.
4. Detection and Identification of Agents of Biological Warfare

5. Detection of Antibiotic Resistance in Bacteria

6. Bacterial Strain Typing and Taxonomy

7. Discrimination strains of beta-hemolytic Streptococci, and also

for characterization of untypable strains of Streptococci group A

Microorganisms can be identified based on their genus level to

specie level going to their strain level

MECHANISM OF MALDI-TOF MS • Discrimination and typing of mycobacteria, typing multidrugresistant K.

1.The sample is mixed with the matrix , then is allowed to crystallized
pneumonia
2. The sample is hit with the laser. Upon the detection of the laser beam,
• Klebsiella has evolved into multi drug resistant.
there is formation of protonated ions.
APPLICATIONS OF MALDI-TOF IN VIROLOGY AND
3. Protonated ions will be separated on the basis of m/z ratio MYCOLOGY
1. Clinical Virology and Clinical Mycology
• It is seen that small protonated ions are the first and the larger ones
2. Viral Genotyping, Subtyping, and Epidemiological Studies
are the last to come out, separated by the m/z ratio.
3. Fungal Strain Typing and Taxonomy
4. TOF would be determined as a detector in this process, also related
4. Detection of Antiviral Resistance and Antibiotic Resistance in Fungi
to the detection of a bacteria.
• General application of MALDI-TOF is specifically for the
5. Final product would be based on the PMF matching.
microorganism. Classification, identification of bacteria type, genus
• It depends if where it can be seen, according to the database this will
level, species level and strain level.
determine the specific analyte.
• Also in typing, subtyping, genotyping and epidemiological studies of
• Established database is needed to have a basis in the identification of
microorganisms
the bacteria.
• To detect microbial resistance - important use of MALDI-TOF.
SO HOW ARE BACTERIA CLASSIFIED?
1. Identification of microbes by MALDI-TOF MS is done by either

comparing the PMF of unknown organism with the PMFs contained in

the database

2. By matching the masses of biomarkers of unknown organisms with

the proteome database.

• In PMF matching, the MS spectrum of unknown microbial isolates is

compared with the MS spectra of known microbial isolates contained in

the database.

• With the help of online information, the identity of a microorganism

can be established down to the genus and in many cases, to the

species and strain level.

• This approach is widely used in microbial identification because it is

simple and can be conveniently adopted in a microbial diagnostic

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