BLOTTING TECHNIQUES..........

1. BLOTTING TECHNIQUES
Present by…
CHOVATIYA MANISH M.
M.SC BIOTECHNOLOGY

2.  Blots are techniques for transferring DNA , RNA
and proteins onto a carrier so they can be
separated, and follows the use of a gel
electrophoresis. The Southern blot is used for
transferring DNA, the Northern blot for RNA and
the western blot for PROTEIN.

3. Blotting technique
Southern Blot
It is used to detect DNA.
Northern Blot
It is used to detect RNA.
Western blot
It is used to detect
protein.

4.  Professor Sir Edwin Southern,
Professor of Biochemistry and
Fellow of Trinity developed this
method in 1975.
 Southern won the Lasker
Award for Clinical Medical
Research prize for the method
of finding specific DNA
sequences he developed this
procedure at Edinburgh
University more than 30 years
ago. The technique is known
as DNA transfer or 'Southern
blotting'
Professor Sir Edwin Southern

5.  This method Involves separation, transfer and
hybridization.
 It is a method routinely used in molecular biology for
detection of a specific DNA sequence in DNA samples.
 The DNA detected can be a single gene, or it can be part of
a larger piece of DNA such as a viral genome.

6.  Southern blotting combines agarose gel electrophoresis for
size separation of DNA with methods to transfer the size
separated DNA to a filter membrane for probe
hybridization.
 The key to this method is Hybridization.
 Hybridization - Process of forming a double-stranded
DNA molecule between a single-stranded DNA probe and a
single-stranded target patient DNA.

7. 1. The mixture of molecules is separated.
2. The molecules are immobilized on a MEMBRANE.
3. The probe is added to the membrane to bind to
the molecules.
4. Any unbound probes are then removed.
5. The place where the probe is connected corresponds to
the location of the immobilized target molecule.

8. Cellulose nitrate (nitrocellulose)
 It is produced by treating cellulose with
nitric acid.
 Each glucose unit in the cellulose polymer is
esterified with three nitrate groups, and
these nitrate groups are responsible for both
the negative charge of nitrocellulose at
neutral pH and the unusual flammability of
dry nitrocellulose.

9.  The major improvement has been the introduction of nylon membranes,
which have three advantages over their nitrocellulose counterparts.
 First,nylon membranes are less fragile than nitrocellulose sheets,the latter
tending to crack if handled roughly during Southern blotting,and usually
disintegrating if attempts are made to carry out more than two or three
hybridization analyses with the same blot.
 Nylon membranes cannot be damaged by handling and a single blot
can be rehybridized up to ten times,this limit being due not to eventual
breakage of the membrane but to the gradual loss of the blotted DNA
during repeated hybridizations.
 The second advantage of nylon membranes is that under certain
conditions (a positively charged membrane and an alkaline transfer
buffer) the transferred DNA becomes covalently bound to the membrane
during the transfer process. This is not the case with a nitrocellulose
membrane,which initially binds DNA in a semipermanent
manner,immobilization occurring only when the membrane is baked at
80C.

10.  Transfer onto a positively charged nylon membrane
can therefore reduce the possible loss of DNA that
might occur by leaching through the membrane
during the blotting process;
 it is also quicker,the transfer time being reduced from
18 h to 2 h.
 Finally,nylon membranes efficiently bind DNA
fragments down to 50 bp in length,whereas
nitrocellulose membranes are effective only with
molecules longer than 500 bp.
 Nitrocellulose has not,however,been completely
superseded because it has one significant
advantage compared with nylon membranes: a
reduced amount of background
hybridization,especially with probes that have been
labelled with nonradioactive markers.

11. 1. Digest the DNA with an
appropriate restriction
enzyme.
2.The complex mixture of
fragments is subjected to
gel electrophoresis to
separate the fragments
according to size.

12. The restriction fragments present in the gel are denatured with alkali
TO GET ss DNA (break Hydrogen bond).
To break the DNA molecules in individual bands within the gel into smaller
fragments, because smaller fragments transfer more quickly than larger
ones. This is achieved by soaking the gel in 0.25 molL21 HCl for 30
min,which results in a small amount of depurination – cleavage of the
b-N-glycosidic bond between purine bases (adenine or guanine) and
the sugar component of their nucleotides – which is followed by
decomposition of the sugar structure and breakage of the
polynucleotide chain.

13.  If a nitrocellulose membrane is being
used then the alkali pretreatment is
followed by neutralization of the gel by
soaking in a Tris-salt buffer,this step being
essential because DNA does not bind to
nitrocellulose at a pH of greater than 9.0.

14.  Nitrocellulose membrane soaked in salt solution
is placed over gel. Nucleic acid move from gel
to membrane . a high concentration of salt
(NaCl THAT IS the positive Na+ ions shield the
negative charges on the phosphodiester
backbone AND LOWERS –ve DNA = -ve
membrane repelling each other
 BAKING Leads to fixing of DNA strongly to the
nitocellulose membrane. UV irradiation,which
results in covalent attachment of DNA to a
nylon membrane.

15.  3 METHODS OF TRANSFER : CAPILLARY
 In a capillary system, rate of transfer depends on the size of
the DNA, thickness of the gel, and agarose concentration.
 Upward capillary transfer is slow, the architecture of the blot
crushes the gel and retards diffusion of the DNA. With a high-salt
buffer, it takes appr. 18 hrs to obtain acceptable transfer
of a 15 kb molecule from a 5 mm thick 0.7% agarose gel; with
the same gel 90% of the 1 kb molecules will be transferred in 2
hrs. This problem is partially alleviated by the depurination
step, which breaks larger molecules in to fragments 1 to 2 kb
in length, thereby reducing the time needed for their transfer.
If the gel is thicker than 5 mm or has an agarose
concentration > 1 %, it cannot be assumed that the larger
fragments will have transferred to a sufficient extent after 12
hr.

16. The weight
helps only in
bringing all
Capillary blotting apparatus
surfaces
together. High
wt may break
the gel.

17. First,the membrane is prehybridized in a solution designed to
block the unused DNA binding sites on the membrane
surface. If this step is omitted then the probe will bind
nonspecifically to the surface of the membrane and the
signal resulting from hybridization to the specific restriction
fragment will be difficult if not impossible to identify.
The prehybridization solution therefore contains nonbiological
polymeric compounds such as polyvinylpyrrolidone and/or
biological polymers such as Ficoll (a carbohydrate-based
compound),bovine serum albumin or dried milk. DNA from
an organism unrelated to the one whose DNA has been
blotted can also be used (salmon sperm DNA is a popular
choice).
Prehybridization takes between 15 min and 3 h at
68C,depending on the type of membrane

18.  enough labeled probe DNA is added to hybridize to the
target restriction fragment to produce a clear signal that
can be discerned by the detection system appropriate for
the label carried by the probe.
The second critical factor that must be considered during the
hybridization step is the specificity of the reaction. If the
probe DNA has been carefully chosen then it will contain a
region that is completely complementary to all or a part of
the blotted restriction fragment that is being sought. If this
hybridizing region in the probe is not completely
complementary to the target,then it will at least have a
region of strong similarity so that a stable hybrid can form.
The problem is that the probe also has the potential to
hybridize to any other blotted DNA fragments with which it
has partial complementarity.

19.  Temperature is relevant because the melting
temperature (Tm,the highest temperature at
which the hybrid is stable) of a fully base-paired
hybrid is higher than that for one in
which some base pairs have not formed
because the probe and target DNAs are not
fully complementary. Hybridization Maximum
rate occurs at 20-25°C below the Tm for DNA-DNA
hybrids, 10-15°C below Tm for DNA-RNA
hybrids
 Formation of the desired hybrid,and
destabilization of nonspecific hybrids,can
therefore be achieved by utilizing an
appropriate combination of buffer composition
and hybridization temperature.

20.  The probe hybridizes to the
complementary DNA restriction
fragment.
Excess probe is washed away
and the probe bound to the filter is
detected by autoradiography,
which reveals the DNA fragment
to which the probe hybridized.

24.  Southern blots are used in gene discovery , mapping,
evolution and development studies, diagnostics and
forensics (It is used for DNA fingerprinting, preparation of
RFLP maps)
 identification of the transferred genes in transgenic
individuals, etc.

25.  Southern blots allow investigators to determine the
molecular weight of a restriction fragment and to measure
relative amounts in different samples.
 Southern blot is used to detect the presence of a particular
bit of DNA in a sample
 analyze the genetic patterns which appear in a person's
DNA.

26. Northern blotting is a technique for detection of specific
RNA sequences.
Northern blotting was developed by James Alwine and
George Stark at Stanford University (1979) and was
named such by analogy to Southern blotting

27.  The first is to determine which tissues
express a particular gene, and this can
give some indication of the physiological
function of the encoded protein.
 The second principal reason for measuring
an mRNA is to determine the factors which
regulate the expression of a given gene,
be they nutritional, hormonal, or
environmental.

28.  Northern blotting.
 A second method is the RNase protection
assay, which is generally considered to offer
improved sensitivity.
 The third method utilizes the reverse
transcriptase polymerase chain reaction; this
provides considerable increases in sensitivity
over Northern blotting and the RNase
protection assay, and may be useful for
measuring very low levels (a few copies) of an
mRNA in a tissue.

30.  Then, in a single step reaction, the
nucleases are inactivated and the
remaining probe:target hybrids are
precipitated. These products are
separated on a denaturing
polyacrylamide gel and are visualized by
autoradiography.

32. 1. RNA is isolated from several biological
samples (e.g. various tissues, various
developmental stages of same tissue etc.)
1. Eukaryotic mRNA can then be isolated
through the use of oligo (dT)
cellulose chromatography to isolate only
those RNAs with a poly(A) tail

33. 2.Sample’s are loaded on
gel and the RNA samples
are separated according
to their size on an
agarose gel .
 The resulting gel following
after the electrophoresis
run.

34. 3. The gel is then blotted on a
nylon membrane or a
nitrocellulose filter paper
by creating the
sandwich arrangement.
Nylon membrane with a
positive charge is the most
effective for use in northern
blotting since the negatively
charged nucleic acids have
a high affinity for them.

35.  Once the RNA has been transferred to
the membrane, it is immobilized
through covalent linkage to the
membrane by UV light or heat.

36. 4. The membrane is placed in a dish
containing hybridization buffer
with a labeled probe.
 Thus, it will hybridize to the RNA
on the blot that corresponds to
the sequence of interest.
5. The membrane is washed to
remove unbound probe.

37. 6. The labeled probe is detected
via autoradiography or via a
chemiluminescence reaction (if
a chemically labeled probe is
used). In both cases this
results in the formation of a
dark band on an X-ray film.
 Now the expression patterns of
the sequence of interest in the
different samples can be
compared.

38.  The RNA samples are most commonly
separated on agarose gels
containing formaldehyde as a
denaturing agent for the RNA to limit
secondary structure.
 Polyacrylamide gel electrophoeresis
with urea can also be used in RNA
separation but it is most commonly used
for fragmented RNA or microRNAs

39.  A standard for the study of gene expression at the level of
mRNA (messenger RNA transcripts)
 Detection of mRNA transcript size
 Study RNA degradation
 Study RNA splicing
 Study RNA half-life
 Often used to confirm and check transgenic / knockout
mice (animals)

40.  Western blotting (1981) is an Immunoblotting technique
which rely on the specificity of binding between a protein
of interest and a probe (antibody raised against that
particular protein) to allow detection of the protein of
interest in a mixture of many other similar molecules.
 Detects proteins and estimates their molecular weight.
 Used to detect changes in protein expression.

41.  Tissue preparation
 Samples can be taken from whole tissue or from cell culture. Solid tissues
are first broken down mechanically using a blender (for larger sample
volumes), using a homogenizer (smaller volumes), or by sonication. Cells
may also be broken open by one of the above mechanical methods.
However, 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. Protease and
phosphatase inhibitors are often added to prevent the digestion of the
sample by its own enzymes. Tissue preparation is often done at cold
temperatures to avoid protein denaturing and degradation.
 A combination of biochemical and mechanical techniques –
comprising various types of filtration and centrifugation – can be used to
separate different cell compartments and organelles.

42. 1. After the samples have been prepared, they are separated
by size using SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) .
1. Since the samples have been denatured in gel loading
buffer containing SDS detergent, the protein is uniformly
negatively charged and will now migrate in an electric
field through the gel and towards the positive electrode .

43.  SDS-PAGE ( sodium dodecylsulphate-polyacrylamide
gel electrophoresis)
 The purpose of this method is to separate
proteins according to their size, and no
other physical feature.

45.  Stacking Gel

48.  Transfer of the proteins fractionated by SDS-PAGE
to a solid support membrane (Western blotting)
can be accomplished by electroblotting

49.  In this procedure, a
sandwich of gel and solid
support membrane
(Nitrocellulose or PVDF) is
compressed in a cassette
and immersed in buffer
between two parallel
electrodes.
 A current is passed at right
angles to the gel, which
causes the separated
proteins to electrophorese
out of the gel and onto the
solid support membrane

50.  The membrane supports used in Western blotting have a
high affinity for proteins. Therefore, after the transfer of the
proteins from the gel, it is important to block the remaining
surface of the membrane to prevent nonspecific binding
of the detection antibodies during subsequent steps.
 A variety of blocking buffers ranging from milk or normal
serum to highly purified proteins have been used to block
free sites on a membrane. The blocking buffer should
improve the sensitivity of the assay by reducing
background interference and improving the signal to
noise ratio. No single blocking agent is ideal for every
occasion since each antibody-antigen pair has unique
characteristics. For true optimization, empirical testing of
blocking buffers is essential.

51.  The balance of SDS in the transfer buffer,
protein size, and gel percentage are the
main factors that affect transfer efficiency.
 About the current and transfer time for
western blot, it's critical to choose the
appropriate current and transfer time for a
successful western blotting.
 Too low current or/and transfer time will
lead to incomplete transfer; if the current
or/and transfer time is too high, on the
contrary, the proteins may migrate
through the membrane too fast without
being absorbed.

52.  One of the critical features of any
successful Western blot is the highly
 specific interaction between an antibody
and an antigen. The antigen,
 usually a protein or peptide, is the target of
the antibody. The precise point
 of interaction is between a small region of
the antigen, an epitope,
 and the recognition sites found on the arms
of the antibody molecule.

55. 1.The confirmatory HIV test
2.Western blot is also used as the definitive test for
Bovine spongiform encephalopathy (BSE(
3.Some forms of Lyme disease testing employ Western
blotting .