Pharmaceutical Biotechnology, BP605T, Western blotting, Southern blotting, ELISA, Immunoblotting techniques, nitrocellulose membrane, horse-radish peroxidase, radioisotope, autoradiography, enzyme-substrate reaction, gel electrophoresis, Bovine serum albumin, preparation of gel for gel electrophoresis, TAE, TBE, SDS-PAGE electrophoresis, blotting techniques, polyacrylamide gel, PVDF, TBS-T, chemiluminescence, Antigen, antibody, spectrophotometer

1. Presented by:-
STEFFI THOMAS
Assistant Professor
School of Pharmacy, LNCTU

2.  Gel electrophoresis is a technique commonly used
in laboratories to separate charged molecules
like DNA, RNA and proteins according to their
size.
 Charged molecules move through a gel when an
electric current is passed across it.
 An electric current is applied across the gel so
that one end of the gel has a positive charge and
the other end has a negative charge.

3.  The movement of charged molecules is called
migration. Molecules migrate towards the
opposite charge. A molecule with a negative
charge will therefore be pulled towards the
positive end (opposites attract!).
 The gel consists of a permeable matrix, a bit
like a sieve, through which molecules can
travel when an electric current is passed
across it.
 Smaller molecules migrate through the gel
more quickly and therefore travel further than
larger fragments that migrate more slowly
and therefore will travel a shorter distance. As
a result the molecules are separated by size.

4.  Electrophoresis enables you to distinguish DNA
fragments of different lengths.
 DNA is negatively charged, therefore, when an
electric current is applied to the gel, DNA will migrate
towards the positively charged electrode.
 Shorter strands of DNA move more quickly through
the gel than longer strands resulting in the fragments
being arranged in order of size.
 The use of dyes, fluorescent tags or radioactive labels
enables the DNA on the gel to be seen after they have
been separated. They will appear as bands on the gel.
 A DNA marker with fragments of known lengths is
usually run through the gel at the same time as the
samples.
 By comparing the bands of the DNA samples with
those from the DNA marker, you can work out the
approximate length of the DNA fragments in the
samples.

5. PREPARATION OF GEL:-
 Agarose gels are typically used to visualise fragments of
DNA. The concentration of agarose used to make the gel
depends on the size of the DNA fragments you are
working with.
 Agarose gels are used for the electrophoresis
of DNA, RNA and Protein. It has got a Horizontal gel
apparatus.
 The higher the agarose concentration, the denser the
matrix and vice versa. Smaller fragments of DNA are
separated on higher concentrations of agarose whilst
larger molecules require a lower concentration of agarose.
 To make a gel, agarose powder is mixed with an
electrophoresis buffer (TAE- (Tris-acetate-EDTA) or TBE-
(Tris-borate-EDTA))and heated to a high temperature
until all of the agarose powder has melted.
 The molten gel is then poured into a gel casting tray and a
“comb” is placed at one end to make wells for the sample
to be pipetted into.

9.  Once the gel has cooled and solidified (it will now
be opaque rather than clear) the comb is
removed.
 Many people now use pre-made gels.
 The gel is then placed into an electrophoresis
tank and electrophoresis buffer is poured into
the tank until the surface of the gel is covered.
The buffer conducts the electric current. The type
of buffer used depends on the approximate size
of the DNA fragments in the sample.
 A dye is added to the sample of DNA prior to
electrophoresis to increase the viscosity of the
sample which will prevent it from floating out of
the wells and so that the migration of the sample
through the gel can be seen.

10.  The prepared DNA samples are then pipetted into
the remaining wells of the gel.
 When this is done the lid is placed on the
electrophoresis tank making sure that the
orientation of the gel and positive and negative
electrodes is correct (we want the DNA to migrate
across the gel to the positive end).
 The electrical current is then turned on so that
the negatively charged DNA moves through the
gel towards the positive side of the gel.
 Shorter lengths of DNA move faster than longer
lengths so move further in the time the current is
run.
 The distance the DNA has migrated in the gel can
be judged visually by monitoring the migration of
the loading buffer dye.

12.  SDS-PAGE Electrophoresis:-
-Sodium Dodecyl Sulfate (SDS) -polyacrylamide
gel electrophoresis is routinely used for the
separation of proteins on the basis of their mass.
-It involves the use of vertical gel apparatus to
separate proteins.
-Buffer system the separation and migration
patterns of proteins in gel electrophoresis are
determined by the chemical composition and pH of
the buffer system.

14.  Blot means a dark mark/stain
 It is a procedure in which a protein or nucleic acid
is separated on a gel are then transferred directly
to an immobilizing medium for identification.
 Blots are techniques for transferring DNA , RNA
and proteins onto a carrier so they can be
separated, and often follows the use of a gel
electrophoresis.

17.  A technique for detecting specific proteins in a
given sample of tissue homogenate or extract
which is separated by electrophoresis by use of
labeled antibodies.
 So called since it has some similarity to a
Southern blot.

18.  Protein blotting is an analytical method that
involves the immobilization of proteins on
membranes before detection using
monoclonal or polyclonal antibodies

19.  Western blotting is an Immunoblotting technique
which rely on the specificity of binding between a
molecule of interest and a probe to allow detection
of the molecule of interest in a mixture of many
other similar molecules.
 In Western blotting, the molecule of interest is a
protein and the probe is typically an antibody raised
against that particular protein.
 The SDS PAGE (Sodium Dodecyl/lauryl Sulfate -
Polyacrylamide Gel Electrophoresis) technique is a
prerequisite for Western blotting .

20.  SDS (has long aliphatic chain + sulfate group)-
is a protein separator i.e. it is an anionic
detergent.
(1)It denatures proteins.
(2)It is used in denaturing polyacrylamide gel
electrophoresis for the determination of protein
molecular weight.
(3)This detergent interacts with denatured
protein to form strong negatively charged
complex.

21.  Western blot analysis can analyze any protein
sample whether from cells or tissues, but also
can analyze recombinant proteins
synthesized in vitro.
 Western blot is dependent on the quality of
antibody you use to probe for your protein of
interest, and how specific it is for this protein
 This method is used in the fields of molecular
biology, biochemistry, immunogenetics and
other molecular biology disciplines.

22.  Western blotting is a more specific test for
detection of HIV.
 It can detect one protein in a mixture of
proteins while giving information about the
size of the protein and so is more specific.
 Western blot test is referred to as the Gold
Standard
 It also tells you how much protein has
accumulated in cells.

23.  If a protein is degraded quickly, Western
blotting won’t detect it well.
 This test takes longer than other existing
tests.
 It might also be more costly

24.  Tissue preparation
 Gel electrophoresis
 Transfer
 Blocking
 Detection
 Analysis

26.  We will introduce an antibody to the target
protein (particular protein) in electrophoresis
so that the interaction occurs.
 If we use 1 antibody it is called primary
antibody that binds to target protein.
 If we use 2 antibodies, it is called secondary
antibody that binds to the primary antibody.

27.  Samples may be taken from whole tissue or from
cell culture.
 In most cases, solid tissues are first broken down
mechanically using a blender. It should be noted
that bacteria, virus or environmental samples can
be the source of protein and thus Western blotting
is not restricted to cellular studies only.
 Assorted detergents, salts, and buffers may be
employed to encourage lysis of cells and to
solubilize proteins.
 Tissue preparation is often done at cold
temperatures to avoid protein denaturing.

28. Resected tissue in pre-cooled (4ºC) normal saline to
wash off any blood from the tissue
Chop the tissue into small pieces (0.1-1g each) after
weighing
Add protease enzyme
Mince the tissue and place the minced tissue in the
tissue homogenizer. Grind the tissue until fully
homogenized.
Add ice-cold lysis buffer (e.g. for 5 mg piece of tissue,
add 300μl of buffer. Buffer volume should be
determined in relation to the amount of tissue present)

29. After ultrasonication, this forms “Tissue
homogenate”
Lyse it on ice for 4-5 hours or at 4ºC (high speed)
for 5 min
Sonication
Centrifugation at 10000 rpm at 4ºC for 10 min
Discard lipid (at top) and cell debris (at bottom).
Take out protein solution in middle and put it in a
fresh tube

30. Mix the protein solution with SDS-PAGE loading
buffer
Denature the solution in 100ºC water bath for 5
min. The solution can be used immediately or
stored at -20ºC for several months or at 4ºC for
1-2 weeks.

31.  The proteins of the sample are separated
using gel electrophoresis. Separation of
proteins may be by isoelectric point
molecular weight, electric charge, or a
combination of these factors.
 The principle involved is the difference in the
ELECTROPHORETIC MOBILITIES of different
proteins.

32.  Uses gel electrophoresis to separate native or
denatured proteins by the length of the
polypeptide (denaturing conditions) or by the
3-D structure of the protein (native/ non-
denaturing conditions).
 The proteins are then transferred to a
membrane (typically nitrocellulose or PVDF),
where they are probed(detected) using
antibodies specific to the target protein.

33.  In order to make the proteins accessible to
antibody detection, they are moved from
within the gel onto a membrane made of
supported nitrocellulose or Polyvinylidene
Difluoride (PVDF).
 So a membrane made up of supported
nitrocellulose (it can withstand autoclave
temperature and retains the ease of wetting
and protein binding features of nitrocellulose)
or PVDF is used.
 PVDF membrane must be wetted in 100%
methanol prior to use, otherwise protein will
not stick onto it due to the presence of SDS.

34.  The membrane is placed on top of the gel,
and a stack of filter papers placed on top of
that. The entire stack is placed in a buffer
solution which moves up the paper by
capillary action, bringing the proteins with it.
Another method for transferring the proteins
is called electroblotting and uses an electric
current to pull proteins from the gel into the
PVDF or supported nitrocellulose membrane.

35.  The membrane has the ability to bind to proteins in
this case both the target and antibodies are proteins
and so there could be some unwanted binding.
 A blocking buffer is a solution of irrelevant protein,
mixture of proteins, or other compound that passively
adsorbs to all remaining binding surfaces of the plate.
It helps in reducing interference by other proteins
other than target protein.
 Blocking of non-specific binding is achieved by placing
the membrane in a dilute solution of protein - typically
TBS-T (Tris-buffered saline with a minute percentage
of detergent such as Tween 20) with Bovine Serum
Albumin (BSA).
 This buffer is used for washing nitrocellulose
membrane in western blotting

36.  The protein in the dilute solution attaches to
the membrane in all places where the target
proteins have not attached. Thus, when the
antibody is added, there is no room on the
membrane for it to attach other than on the
binding sites of the specific target protein
 After this the membrane is incubated with 1º
antibody specific to target protein.

37.  During the detection process, the membrane is
"probed" for the protein of interest with a
modified antibody which is linked to a reporter
enzyme, which when exposed to an appropriate
substrate drives a colorimetric reaction and
produces a color.
 Then the membrane is incubated with HRP-
labelled (Horse Radish Peroxidase is the enzyme
that enhance chemiluminescence) 2º antibody
specific to 1º antibody.
 The point where signal is produced by 2º
antibody, is where the target protein is located.

38.  After the unbound probes are washed away, the
western blot is ready for detection of the probes
that are labeled and bound to the protein of
interest.
 Size approximations are taken by comparing the
stained bands to that of the marker loaded
during electrophoresis.
 Image is produced (black dots and bands are
produced)
 Dark bands- high concentration
 Faint band- lowest concentration

40.  To detect HIV
 To detect BSE (Bovine Spongiform
Encephalopathy) (Mad cow disease)
 Confirmatory test for Hepatitis B infection

41.  A Southern blot is a method used in molecular
biology for detection of a specific DNA sequence
in DNA samples.
 The Southern Blot allows the visualization of one
DNA fragment from a whole genome DNA
extract.
 Southern blotting combines transfer of
electrophoresis -separated DNA fragments to a
filter membrane and subsequent fragment
detection by probe hybridization.
 The method is named after its inventor, the
British biologist Edwin Mellor Southern.

42.  The key to this method is hybridization.
 Hybridization: It is the process of forming a
double-stranded DNA molecule between a
single-stranded DNA probe and a single-
stranded target DNA.
 There are 2 important features of hybridization:
• The reactions are specific-the probes will only
bind to targets with a complementary sequence.
• The probe can find one molecule of target in a
mixture of millions of related but non-
complementary molecules.

43.  DNA-DNA hybridization:- Uses gel
electrophoresis together with hybridization
probes to characterize restriction fragments
of genomic DNA (or DNA from other sources,
such as plasmids)

45. Step 1:-Extract and purify DNA from cells
•Isolate the DNA from the rest of the cellular
material in the nucleus.
•Incubate specimen with detergent to promote cell
lysis.
•Lysis frees cellular proteins and DNA.
•Proteins are enzymatically degraded by
incubation with proteinase.
•Organic or non-inorganic extraction removes
proteins.
•DNA is purified from solution by alcohol
precipitation.
•Visible DNA fibers are removed and suspended in
buffer. DNA is restricted with enzymes
(restriction endonuclease).

46. Step 2:-Gel Electrophoresis
• Separates DNA fragments according to size
• Denature DNA-
- The restriction fragments present in the gel
are denatured with alkali.
-This causes the double stranded to become
single-stranded.
- DNA is then neutralized with NaCl to
prevent re-hybridization before adding the
probe.

48. Step 4:-Hybridization
•Incubate nitrocellulose sheet with a minimal
quantity of solution containing 32P-labeled
ssDNA probe.
•Probe sequence is complementary to the DNA
of interest.
•Incubate for several hours so that probe can
anneal to its target sequence(s).
•Wash & dry nitrocellulose sheet.

49. Step 5:-Autoradiography
•Place nitrocellulose sheet over X- ray film.
•X-ray film darkens where the fragments are
complementary to the radioactive probes.
• If the probe is radioactive, the particles emits
when expose to X-ray film.

50.  Diagnosis and detection of genetic diseases
such as sickle cell anaemia
 Used in forensics (DNA fingerprinting)
 To identify specific DNA in a DNA sample
 Identify mutations, deletions, and gene
rearrangements.
 Prognosis of cancer and in prenatal diagnosis
of genetic diseases.

51.  It is a very sensitive technique to detect the
presence of antigen or antibody in blood.
 It is similar to Radioimmuno Assay (RIA), but it
uses radioisotopes to see the presence of
antigen or antibody.
 In case of ELISA we use enzyme-substrate
reaction and the colouration produced due to it
(i.e. enzyme-substrate reaction) as a process of
identifying antigen or antibody.

52.  Antigen/antibody is adsorbed on to the plastic
surface (‘sorbent’)
 Antigen is recognized by specific antibody
(‘immuno’)
 This antibody is recognized by secondary
antibody which has an enzyme attached
(‘enzyme-linked’)
 Substrate reacts with the enzyme to produce a
product, usually coloured.

53.  As it is a very sensitive technique, it can
detect protein even if it in picogram level.
 For e.g. if you have an infection (antigen) in
your body due to any bacteria, fungi or virus,
your body produces antibody which is
specific against that particular infection. If we
determine that particular antibody in your
body, this means that there is infection in
your blood serum.

54. radish

56.  Enzyme: Horse Radish Peroxidase (HRP), MW 44,
000, glycoprotein with 4 lysine residues.
 Substrate: TMB (3,3',5,5', tetramethylbenzidine).
The enzyme acts as a catalyst to oxidize substrate
in the presence of Hydrogen peroxide to produce a
blue color. Reaction stopped with dilute acid to
cause complex to turn yellow.

57.  96 wells Microtiter plate (8×12)

58.  Multipipette (8 channel 100μl pipette)

59. radish

60. 1.Antibody is coated inside
the well
2.Antigen containing
solution is added which
binds to the antibody, after
this washing is done to
remove unbound antigens
to be washed away
3.An antibody (2º antibody)
specific to that antigen is
added which is linked with
an enzyme (depicted in
circular red dots)
4.Substrate (Yellow
coloured circle) is added,
which reacts with enzyme
to form a coloured poduct

62.  INDIRECT ELISA
 COMPETITIVE ELISA
 SANDWICH ELISA

64.  Antibody can be detected or quantitatively
determined with Indirect ELISA
 Serum or some other sample containing primary
antibody (Ab1) to an antigen-containing microtiter
well and allowed to react with the antigen attached
to the well.
 After any free Ab1 is washed away, the presence of
antibody bound to the antigen is detected by adding
an enzyme-conjugated secondary antibody (Ab2),
which binds to primary antibody.
 Any free Ab2 then is washed away and a substrate
for the enzyme is added.
 The amount of coloured reaction product formed is
measured by specialized spectrophotometric
methods.
 It is so called because the enzyme is linked to
secondary antibody and not the primary antibody.

66.  Another variation for measuring amount of
antigen is competitive ELISA.
 In this technique, antibody is first incubated in
solution with a sample containing antigen.
 The antigen-antibody mixture is then added to
an antigen coated microtiter well.
 The more antigen present in the sample, the
less free antibody will be available to bind to the
antigen-coated well.
 Addition of an enzyme-conjugated secondary
antibdy (Ab2) specific for the primary antibody
can be used to determine the amount of primary
antibody bound to the well as in indirect ELISA.

68.  Antigen can be detected or measured by
sandwich ELISA.
 Here antibody (rather than antigen) is
immobilized on microtiter well.
 A sample containing antigen is added and
allowed to react with the immobilized antibody.
After this the well is washed
 A second enzyme-linked antibody specific for a
different epitope on the antigen is added and
allowed to react with the bound antigen.
 After any free second antibody is removed by
washing, substrate is added, and the colored
reaction product is measured.

70.  https://microbeonline.com/western-blot-
technique-principle-procedures-advantages-
and-disadvantages/
 Punt Jenni, Stranford Sharon, “Kuby
Immunology”, Published by W.H. Freeman &
Co Ltd., 8th edition, Pg no. 148-154
 https://www.slideshare.net/sabaahmed56/eli
sa-ppt-53678570