Blotting techniques allow for the detection of specific biomolecules like DNA, RNA, and proteins. Southern blotting detects DNA through electrophoresis, transfer to nitrocellulose, and hybridization with a radioactively labeled probe. Northern blotting is similar but detects RNA. Western blotting detects proteins through electrophoresis, transfer, and incubation with labeled antibodies. These techniques are used for applications like gene mapping, fingerprinting, and confirming infections.
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes to investigate the presence of small, submicroscopic chromosomal changes that are beyond the resolution of karyotype analysis.
This PowerPoint presentation explain the concept,process and application of Fluorescence insitu hybridization.
Principle: The method is characterized by transferring the protein which run on a gel by electrophoresis on to a nitro cellulose membrane.
It is widely used analytical technique in molecular biology to detect specific proteins in a sample of tissue homogenate or extracts.
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes to investigate the presence of small, submicroscopic chromosomal changes that are beyond the resolution of karyotype analysis.
This PowerPoint presentation explain the concept,process and application of Fluorescence insitu hybridization.
Principle: The method is characterized by transferring the protein which run on a gel by electrophoresis on to a nitro cellulose membrane.
It is widely used analytical technique in molecular biology to detect specific proteins in a sample of tissue homogenate or extracts.
Blotting technique including Southern , Northern and Western blotting Rohit Mondal
he given ppt contains all the blotting techniques which is being studied by students in Biotechnology related subject and this PPT contais all blotting techniques in a very elaborative concise manner includes procedure principle application etc so which itwould help any bio student to take proper knowledge in this topic. I hope you will enjoy the content of the topic and would be able to grasp the topic properly
WHAT IS BLOTTING?
Blotting is a technique for detecting any macromolecules that we deal with like DNA, RNA or proteins, which are initially present in a complex mixture.
TYPES OF BLOTTING:
Southern Blotting
Northern Blotting
Western Blotting
NORTHERN BLOTTING
A northern blotting is a laboratory method used to detect specific RNA molecules among a mixture of RNA (mRNA).
The technique was developed in 1979 by James Alwine and his colleagues.
Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell type in order to measure the expression of particular genes.
Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence.
The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However the entire process is commonly referred to as northern blotting.
PROCEDURE
1.RNA isolation:
2.Separation of RNA using gel electrophoresis:
3.BLOTTING:
4.Hybridization with labelled probe:
5.WASHING OFF EXCESS PROBES
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The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
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2. What is Blotting
Blotting is a powerful and sensitive technique for identifying
the presence of specific biomolecules within a sample.
Subtypes of blotting are differentiated by the target
molecule that is being sought.
3. Types of blotting
Southern blotting
( to detect DNA )01
Northern blotting
( to detect RNA )
02
03 Western blotting
( to detect protein )
4. SOUTHERN BLOTTING
Southern blotting is the first blotting technique which made
analysis and recording easy.
In Southern blotting ,a DNA fragment containing a specific
sequence can be identified by electrophoresis , transferring
them into nitrocellulose & hybridizing with a 32p labelled
single stranded DNA probe complementary to the sequence.
The fragment containing the sequence then visualized by
audioradiography
It was first developed by E.M.Southern in 1975
6. PROCEDURE
For Southern blotting , DNA sample is first digested with a
restriction enzyme and digested sample is electrophoresed.
The DNA bands in the gel are denatured into single strands with the
help of an alkali solution.
Subsequently the gel is laid on top of a buffer saturated filter paper
placed on a solid support(eg.glass plate),with its two edges immersed
in the buffer.
A sheet of nitrocellulose filter membrane is placed on top of the
gel and a stack of many papers on top of this membrane.
7. Cont…
01
02
Capillary action pulls the buffer from the bottom
filter through the gel, to the transfer medium
and up through the paper towel stack.
While passing through the gel, the buffer carries
with its single stranded DNA, which binds on to
the nitrocellulose membrane, when the buffer
passes through it to the paper towels.
The nitro cellulose membrane with single
stranded DNA bands blotted on to it, is baked
at 80c for 2-3 hours to fix the DNA permanently
on the membrane.
DNA fragments on membrane can then be probed
for sequence of interest by hybridization between
them.
Membrane is washed to remove the unbound DNA
X-ray film is then exposed to the
membrane to get
autoradiographs.
8. Applications
Southern blotting is extremely
sensitive and can be applied to
mapping restriction sites around a
single copy gene sequence in a
complex genome such as that of
man.
When mini satellite probe is used
the technique can be also used for
forensic purpose for identification of
minute amount of DNA.
The use of southern blot
technique has also been
done for analyzing the role
of DNA methylation in gene
expression.
Blotting analysis useful in
detection of mutated genes
in genetic disorders.
9. Restriction analysis of genes studied with southern blot helps in this respect.
DNA finger printing. RFLP mapping.
10. Northern Blotting
The Northern blot procedure essentially identical to
that of southern blotting except that here RNAs are
separated by gel electrophoresis.
Initially southern blotting could not be used
directly to blot transfer RNA from gel to
nitrocellulose membrane because RNA did not
bind to cellulose nitrate.
Alwine,Kemp and Stark(1979)developed a
procedure for blot transfer of RNA.
He used a chemically reactive paper(diazotization of
amino benzyl oxymethyl paper which is prepared from
Whatsman 540 papers).
11.
12. PROCEDURE
1. RNA species are separated on
the basis of size by
electrophoresis through
agarose gel containing
formaldehyde or glyoxal and
dimethyl sulfoxide.
2. The separated RNA bands are then
blotted on chemically reactive filter
paper.
3. RNA species after blotting are
hybridized to radiolabelled DNA
probe.
4. Auto radiography is carried onto
locate RNA bands that are
complementary to the probe.
13. APPLICATIONS
Used for detection and
quantitative estimation of
hybridized mRNA.
Study RNA degradation
Study RNA half life
Study RNA splicing
It is useful in the studies of gene
expression
14. WESTERN
BLOTTING
This technique is used to detect the proteins of a
particular specificity.
When a transferred gene expresses in transformed
cells,
The translated product in the form of proteins can
be identified by this technique.
15. PROCEDURE
40% 50%60%80%
First we isolate the protein or extract the protein.
The extracted proteins are subjected to
PAGE(Poly Acrylamide Gel Electrophoresis)
and are then transferred onto nitro cellulose to
which they bind.
Then radiolabelled specific antibody is added on
such membrane and it binds only to specific
complementary protein.
The antibody is labelled with 125 I and the
signal is detected again with autoradiography.
If radio active label is not used, bound antibody may
be detected by a second antibody tagged with an
enzyme.
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17. APPLICATIONS
04
05
06
01
02
03
The confirmatory HIV test employs a western blot to detect
anti- HIV antibody in a human serum sample. Proteins
from known HIV-infected cells are separated and blotted
on a membrane as above. Then, the serum to be tested is
applied in the primary antibody incubation step; free
antibody is washed away, and a secondary anti-human
antibody linked to an enzyme signal is added. The stained
bands then indicate the proteins to which the patient's
serum contains antibody.
A western blot is also used as the definitive test
for Bovine spongiform encephalopathy (BSE,
commonly referred to as 'mad cow disease').Western blot can also be used as a
confirmatory test for Hepatitis B infection
