Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow for the detection of specific DNA, RNA, and protein sequences by transferring them from a separation gel onto a membrane and using probes to detect the targets. Southern blotting detects DNA using DNA probes, Northern blotting detects RNA using RNA or DNA probes, and Western blotting detects proteins using antibodies. These techniques are used for applications like detecting gene expression, RNA splicing, and confirming diseases.
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
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
Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
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.
Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
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.
What is blotting? 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. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN.
What is teaching methodology, Objectives, Parts of teaching methodologies, Types of Teaching methods, Lecture method, Basic feautres , Purpose of these methods, Advantages and Disadvantages, Limitation of teaching methods, Team teaching method, Steps of team teaching methods, Characterstics of teaching methods, TV or Video Presentations, Group discussion method, Kinds of team teaching, Discussion methods of learning, Seminar method, Advantages and Disadvntages of seminar method, Brainstorming, Advantages and Disadvantages of Brain storming, Project method, Strategy of Project based teaching strategy, Characterstics of Project method, Role of teacher, Merits and Demerits of Project method.
INTRODUCTION OF COVID-19, ORIGIN OF COVID-19, STRUCTURE OF COVID-19, CAUSES OF CORON VIRUS, SYMPTOMS OF COVID-19, TYPICAL SYMPTOMS OF COVID-19, MODE OF TRANSMISSION, PEOPLE WHI ARE AT HIGHER RISK, WHY COVID-19 IS SAID T BE AS THE PANDEMIC BY WHO?, PREVENTION, WHAT TO DO, WHAT NOT TO DO, MYTHS AND FACTS OF COVID-19 SPREADING, SOME OTHER CONSEQUENCES OF COVID-19, MOST IMPORTANT POINTS OF COVID-19, COVID-19 VACCINES INTRODUCTION, TYPES OF VACCINES , COVAXIN, COVISHIELD, COVID VACCINE REGISTERATION, WHO CAN REGISTER, WHO SHOULDNT TAKE VACCINE SHOTS, STEP BY STEP GUIDE FOR REGISTERATION, COMPARISON BETWEEN COVAXIN AND COVISHIELD,
WHAT IS PHOTOSYNTHESIS?, IMPORTANCE OF PHOTOSYNTHESIS, STRUCTURAL FEATURE OF LEAF ADVANTAGE FOR PHOTOSYNTHESIS,LEAVES AND LEAF STRUCTURE,CHLOROPHYLL, TYPES OF REACTIONS, LIGHT REACTION AND DARK REACTION, CYCLIC AND NON-CYCLIC PHOTOPHOSPORYLATION, MECAHANISM OF ATP SYNTHESIS, SCHEMATIC PRESENTATION OF LIGHT REACTION, CRASSULACEAN ACID METABOLISM (CAM), C3 AND C4 PLANTS, FACTORS AFFECTING RATE OF PHOTOSYNTHESIS, INTERNAL FACTORS AND EXTERNAL FACTORS,
RECOMBINANT DNA GUIDELINES DEFINATION, RESEARCH ACTIVITIES AND ITS CATEGORIES,BIOSAFETY LEVELS, BSL-1, BSL-II, BSL-III, BSL-IV, WHAT IS BIOSAFETY GUIDELINES, AIM OF BIOSAFETY GUIDELINES, THE R-DNA BIOSAFETY GUIDELINES IN INDIA , COMMITTEES IMPANTED BY DBT, IBSC, ECGM, GEAC, CONTAINMENTS AND ITS TYPES, LEVELS OF CONTAINMENTS,PURPOSE OF THE CONTAINMENTS, ELEMENT OF CONTAINMENTS, IMPLEMENTATION OF BIOSAFETY GUIDELINES,MECHANISM OF IMPLEMENTATION, PHYSICAL CONTAINMENTS, BIOLOGICAL CONTAINMENTS, IMPLEMENTATION OF BIOSAFTEY GUIDELINES, RECOMBINANT DNA ADVISORY COMMITTEE, INSTITUTIONAL BIOSAFETY COMMITTEE,
Site directed mutgenesis, OLIGONUCLEOTIDE DIRECTED MUTAGENESIS Vipin Shukla
INTRODUCTION, HISTORY, MUTATION, DIRECTED MUTAGENESIS,BASIC MECHANISM OF SITE DIRECTED MUTAGENESIS,METHOD FOR SITE DIRECTED MUTATIONS,THE SINGLE PRIMER METHOD, CASETTEE MUTAGENESIS, PCR-SITED DIRECTED MUTAGENESIS, APPLICATION OF SITE DIRECTED MUTAGENESIS.
INTRODUCTION, DEFINATION OF ELECTROPHORESIS, ELECTROPHORESIS PRINCIPLE, TYPES OF ELECTROPHORESIS, FREE ELECTROPHORESIS, ZONE ELECTROPHORESIS,PAPER ELECTROPHORESIS, WORKING OF PAPER ELECTROPHORESIS, PROCEDURE FOR PAPER ELECTROPHORESIS, VISUALISATION, FACTORS AFFECTING SEPARATION OF MOLECULES, APPLICATIONS, working of paper electrophoresis ,procedure for paper electrophoresis ,visualisation ,factors affecting separation of molecules ,applications ,forensics ,dna fingerprinting ,molecular biology ,microbiology information about the organisms ,biochemistry mapping of cellular components ,paper electrophoresis is also used in study of sic ,hemoglobin abnormalities ,separation of blood clotting factors ,serum plasma proteins from blood sample ,used in separation and identification of alkaloids ,used for testing water samples ,toxicity of water ,drug industry to determine presence of illelgal drUGS
Introduction, Theme of the environment day, Role of teachers in environmental education, How the environment impact our health, How can we celebrate the day, Dangerous or Hazardous Waste, Seven Billion Dreams, Our planet consume with care, Examples such as Radioactivity wastes, Soil erosion
Reproductive system and its Classification Vipin Shukla
Human Reproductive system, Classificatio of Human reprodutive system, Parts and Functions male reproductive organs, Female Reproductive system, Parts and functions of female reproductive system,The Menstrual cycle, Assisted Reproductive Technology, Invitrofertilization, and its techniques, Most commonly used techniques, Les commonly used techniques, Transvaginal Oocytes Retrieval, Intra Cytoplasmic Injecection, (ICSI), Procedure of ICSI, Who are the patients required ICSI, Embryo Transfer, Zygote Intra Fallopain Transfer, Gametes Intrafallopian Transfer, Gift Technique, Surrogacy, Types of Surrogacy, Steps Involved in Surrogacy, Ither techniques,
Human excretion is the process of removing excess water, waste materail and harmful substances from human body.
Also excretion is the process of eliminationg waste products of metabolism and other non-useful materials.
It is an essintial process in all formes of life. It also eliminate waste products such as water, carbondioxide and nitrogenous wates formed during catabolism.
Revised guideline for research in transgenic plants (Vipin Shukla
In 1998, BDT brought out seperate guidelines for carriying out research in transgenic planst called the Revised Guidelines for research guidelines in Transgenic Plants.
Approaches of biotechnology in medicalVipin Shukla
Medical Biotechnology is defined as the branch of science that delas with the study of use of living cells in Research and pharmaceuticals and diagnostic products that help to treat and prevent human diseases.
Bio saftey in transgenics & its productsVipin Shukla
Transgenic plants are those plants were we insert an foreign gene in an host genome to modify its characters such as Stress tolerance, Virus resistant, Biotic and Abiotic Tolerance etc.
Polymerase Chin Reaction is a technique that takes specific sequences of DNA of small and amplifies it to be used for further testing.
it is also said to be as the Invitro Technique.We have seen an photocopy machine in an office, by which we can copy several pages. So, is the PCR machine in a molecular biology laboratory.
PCR is DNA raplication ina test tube.
Dr Kary Mullis developed PCR.
To amplify lot of double stranded DNA molecules with same size and sequence by enzymatic method and cycling condition.
Biosaftey means the needs to protect human and animal health along with the environment from the possible adverse effects of the products of modern biotechnology. Biosafety defines the containment conditions under which infectious agents can be safely manipulated. Biosafety word is used to reduce and eliminate the potential risk regulating from the modern biotechnology and its products.
HYBRIDOMA TECHNOLOGY IT IS DEFINED AS THE PROCESS WERE THERE IS A FUSION OF SPLLEN CELL AND MYELOMA CELLS IN THE PRESENCE OF POLYETHYLENE GLYCOL OR SENDAI VIRUS AND LEADS TO THE PRODUCTION OF MONOCLONL ANTIBODY.
COMPLEMENT SYSTEM IS DEFINED AS THE PART OF IMMUNE SYSTEM WHICH ENHANCES THE IMMUNITY OF AN INDIVIUAL. IT INCLUDES 30 SOLUBLE PROTEINS PRESENT IN PLASMA.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
1. PREPARED BY: VIPIN KUMAR SHUKLA
ASSISTANT PROFESSOR
DEPARTMENT OF BIOTECHNOLOGY
BLOTTING & ITS TYPES
2. What is blotting?
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.
The Southern blot is used for transferring DNA, the
Northern blot for RNA and the western blot for
PROTEIN.
3. TYPES OF BLOTTING TECHNIQUES:
Northern
Blot
It is used to
detect
RNA.
Southern Blot
It is used to
detect
DNA.
Blotting
Technique
Western blot
It is used to
detect
PROTEIN
4. SOUTHERN BLOTTING:
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
5. Continued……
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. Continued…….
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. PRINCIPLE:
The mixture of molecules is separated.
The molecules are immobilized on a matrix.
The probe is added to the matrix to bind to the
molecules.
Any unbound probes are then removed.
The place where the probe is connected corresponds
to the location of the immobilized target molecule.
13. Continued…..
Digest the DNA with an
appropriate restriction
enzyme.
The complex mixture of
fragments is subjected to gel
electrophoresis to separate the
fragments according to size.
14. Continued…..
The restriction fragments present
in the gel are denatured with
alkali and transferred onto
A nitrocellulose filter or nylon
membrane by blotting.
This procedure preserves the
distribution of the fragments in
the gel, creating a replica of the
gel on the filter.
15. Continued….
The filter is incubated under
hybridization conditions with a
specific radio labeled DNA probe.
The probe hybridizes to the
complementary DNA restriction
fragment .
16. Continued…..
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.
18. Application:
To determine the conditions under which specific
genes are being expressed(M-rna level).
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
Often used to confirm and check transgenic/knock out
mice(animals).
19. Northern Blotting:
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.
20. Steps involved in Northern blotting:
RNA is isolated from several
biological samples (e.g. various
tissues, various developmental
stages of same tissue etc.)
RNA is more susceptible to
degradation than DNA.
SS-RNA can form the secondary
structure during running.
Use formaldehyde to break H
bonds and denature RNA because
single-stranded RNA will form
intramolecular base pairs
and"fold"onitself.
21. Continued……
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.
22. Continued…..
The gel is then blotted
on a nylon membrane
or a nitrocellulose filter
paper by creating the
sandwich arrangement.
23. Continued…..
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.
The membrane is
washed to remove
unbound probe.
24. 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.
25. APPLICATIONS:
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)
26. Disadvantage of Northern blotting:
The standard northern blot method is relatively less
sensitive than nuclease protection assays and RT-
PCR Detection with multiple probes is a problem.
If RNA samples are even slightly degraded by
RNases, the quality of the data and quantitation of
expression is quite negatively affected.
27. Western blotting:
Western blotting (1981) is an
Immuno blotting 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.
The SDS PAGE technique is a
prerequisite for Western blotting .
28. Steps in western blotting:
A protein sample is
subjected to
electrophoresis on an
SDS polyacrylamide gel.
Electro blotting transfers
the separated proteins
from the gel to the
surface of a nitrocellulose
membrane
29. Continued……
The blot is incubated with a generic
protein (such as milk proteins or
BSA) which binds to any remaining
sticky places on the nitrocellulose.
An antibody that is specific for the
protein of interest (the primary
antibody - Ab1) is added to the
nitrocellulose sheet and reacts with
the antigen. Only the band containing
the protein of interest binds the
antibody, forming a layer of antibody
molecules .
30. Continued……
After washing for removal of non-
specifically bound Ab1, second
antibody (Ab2)is added, which
specifically recognizes the Fc
domain of the primary antibody and
binds it.
Ab2 is radioactively labeled, or is
covalently linked to a reporter
enzyme, which allows to visualize
the protein-Ab1-Ab2 complex.
31. Application:
The confirmatory HIV test
Western blot is also used as the definitive test for
Bovine spongiform encephalopathy (BSE(
Some forms of Lyme disease testing employ
Western blotting
32. Comparison of Southern, Northern,
and Western blotting techniques:
Molecule
detected
Southern
blotting
Northern
blotting
Western blotting
Gel
electrophoresis
Agarose gel Formaldehyde
agarose gel
Polyacrylamide
gel
Gel pretreatment Depurination,
Denaturation, &
neutralization
---------- ---------
Blotting method Capillary transfer Capillary transfer Electric transfer
Probes DNA Radioactive
or nonradioactive
cDNA, cRNA
Radioactive or
nonradioactive
primary antibody
Detection system Autoradiography
Chemiluminesce
nt Colorimetric
Autoradiography
Chemiluminesce
nt Colorimetric
Chemiluminesce
nt Colorimetric
33. Application:
The confirmatory HIV test
Used as the definitive test for Bovine spongiform
encephalopathy(BSE).
Some forms of Lyme disease testing employ
Western blotting