''Electrophoretic Mobility Shift Assay'' by KATE, Wisdom DeebekeWisdom Deebeke Kate
This assessed presentation was delivered by me, together with other three course mates. The aim of the presentation was to describe the basic principles, methods involved in EMSA, and some of its application in molecular biology to study the interactions between proteins and DNA. Delivered on 9th December, 2013 with Lolomari Songo, Nicholas Leach & Abhay Jethwani.
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
''Electrophoretic Mobility Shift Assay'' by KATE, Wisdom DeebekeWisdom Deebeke Kate
This assessed presentation was delivered by me, together with other three course mates. The aim of the presentation was to describe the basic principles, methods involved in EMSA, and some of its application in molecular biology to study the interactions between proteins and DNA. Delivered on 9th December, 2013 with Lolomari Songo, Nicholas Leach & Abhay Jethwani.
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
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Blotting
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier.
The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.
Types of blotting techniques
Southern Blotting
Northern Blotting
Western Blotting
A Southern blot is a method used
in molecular biology for detection of a specific DNA sequence in DNA samples.
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.
- Methods in Southern blotting
- Advantages and disadvantages
Southern blotting is a laboratory technique used to detect specific DNA sequences in DNA samples. It involves several steps:
Restriction Digestion: DNA from a biological sample (such as blood or tissue) is broken into smaller fragments using restriction enzymes, which cut the DNA at specific sequences.
Electrophoresis: The DNA fragments are separated based on their molecular weights using gel electrophoresis. This process allows smaller fragments to move faster than larger fragments.
Transfer to Membrane: The DNA fragments are transferred from the gel onto a solid membrane, typically a positively charged nylon membrane, using capillary action.
Hybridization: The membrane is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The probe is designed to be complementary to the target DNA sequence, allowing it to bind to the specific DNA fragment on the membrane.
Detection: The bound probe is detected using methods such as X-ray film, phosphorimaging, or chemiluminescent substrates, depending on the type of label used.
Southern blotting is used in various applications, including:
Identifying specific DNA sequences in DNA samples
Studying gene rearrangements and mutations
Analyzing viral and bacterial infections
Forensic analysis and personal identification
Gene mapping and restriction enzyme mapping
Identifying methylated sites in genes.
This technique is named after its inventor, Dr. Edwin Southern, who first published it in 1975.
This is all about southern blotting
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier (for example, a nitrocellulose, polyvinylidene fluoride (PVDF) or nylon membrane). In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining (for example, silver staining of proteins), autoradiographic visualization of radioactive labelled molecules (performed before the blot), or specific labelling of some proteins or nucleic acids. The latter is done with antibodies or hybridization probes that bind only to some molecules of the blot and have an enzyme joined to them. After proper washing, this enzymatic activity (and so, the molecules we search in the blot) is visualized by incubation with proper reactive, rendering either a colored deposit on the blot or a chemiluminiscent reaction which is registered by photographic film.
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.[1]
Western blot
A Western blot is used for the detection of specific proteins in complex samples. Proteins are first separated by size using electrophoresis before being transferred to an appropriate blotting matrix (usually PVDF or nitrocellulose) and subsequent detection with antibodies.
Eastern blot
The Eastern blot is used for the detection of specific posttranslational modifications of proteins. Proteins are separated by gel electrophoresis before being transferred to a blotting matrix whereupon posttranslational modifications are detected by specific substrates (cholera toxin, concanavalin, phosphomolybdate, etc.) or antibodies.
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.
MICROBIAL SPOILAGE
TYPES OF SPOILAGE
PHARMACEUTICAL SPOILAGE
MICROBIAL SPOILAGE OF PHARMACEUTICALS
TYPES OF MICROBIAL SPOILAGE
REASON OF CONTAMINATION
FACTORS AFFECTING SPOILAGE OF PHARMACEUTICAL PRODUCTS
B pharma
D pharma
Pharmaceutical Biotechnology
Pharmaceutics I
Immunity and Immunological Products
types of immunity
Immunology
Toxins antibody exotoxins endotoxins
Vaccine
toxoids
sera
B.C.G. vaccine.
cholera. pertussis, plague and typhoid vaccine.
typhus vaccine.
measles, small-pox. poliomyelitis and yellow fever.
diphtheria, tetanus and staphylococcus.
Diagnostic preparations containing bacterial toxins used for Schick test and tuberculin test.
Preparations containing antibodies (antiserum, and antitoxins)used to produce passive immunity
Unit 2 organization and personnel and permisies himanshuhimanshu kamboj
pharmaceutical quality assurance
b pharma 6th sem
Personnel objectives
Personnel qualifications
Personnel responsibilities
Key personnel
Responsibilities of the head of the production department
Responsibilities of the head of quality control department
Training
Personnel hygiene
Premises
Layout of pharmaceutical industry
Areas of premises
Environmental control in sterile areas
Equipment and raw materials
Stages of equipment
Cleaning and maintenance
Raw materials
Steps involved in purchase procedure
Maintenance of stores
Storage conditions
B PHARMA 6TH SEM
PHRAMACEUTICAL QUALITY ASSURANCE
Pharmaceutical documentation
Need of documentation
Objectives of documents
Scope
Documentation lifecycle
Types of documents
Characteristic of document
Documentation review
Documents model
Standard operating procedures (sop’s)
Master formula record
Batch formula record
Quality audit plan and reports
Specification and test procedures
Pharmaceutical Microbiology
What is spoilage?
Types of spoilage
Pharmaceutical spoilage
Microbial spoilage of pharmaceuticals
Types of microbial spoilage
Reason of contamination
Factors affecting spoilage of pharmaceutical products
B PHARAM 6TH SEM
PHARAMACEUTICAL QUALITY ASSURANCE
COMPLAINT
Reasons
Types of Complaint
Steps involved in Handling of complaints
Product Complaint Data Sheet
Complaint Record
Regulatory Guidelines
SOP on Complaint Handling
RECALL
Reasons
Types of Recall
Recall Classification
Levels of Recall
How to Recall the Product?
How To Notify The Consumers?
Regulatory Guidelines
SOP on Product Recall
DRUG RECALL IN 2013 AND 2014
Qc test for plastics,metallic tins,closures, collapsible tubes, secondary pac...himanshu kamboj
b pharma 6th sem
pharmaceutical quality assurance
Introduction
Types of pharmaceutical packaging
Packaging materials
Quality control test for plastic
Quality control test for closures
Quality control of collapsible tubes
Quality control of metallic tins
QC test for secondary packaging materials
Protein engineering and its techniques himanshuhimanshu kamboj
b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components
b pharmacy
pharmaceutical biotechnology
Polymerase chain reaction
History
Purpose
Components of PCR
Steps of PCR
Denaturation of DNA template
Annealing of primers
Extension of ds DNA molecules
Reaction Condition & Experimental Protocol
General PCR Protocol
Application
b pharma 6th sem
nucleic acid extraction and quantification
pharmaceutical biotechnology
Introduction
Purpose
Isolation
Methods of isolation
Basic steps for DNA extraction
Organic extraction
Inorganic extraction
salting out
Introduction
Gel Electrophoresis
Principle of separation
Instrument and reagents
Factors affecting separation in gel electrophoresis
Applications
Electrophoresis apparatus
Buffer
Power supply
Supporting media
Detection and Quantification
Agarose
Polyacrylamide
6th sem b pharma QC,QA
pharmaceutical quality assurance
Quality Management System
Introduction to ISO 9000
Eight Quality Management Principles
ISO 9000 Series
Advantages
Clauses
Introduction to ISO 14000
Standards under ISO 14000 series
NABL accreditation ( National Accreditation Board for Testing and Calibration Laboratories)
Function of NABL
Process of NABL
Good Laboratory Practices (GLP)
History
Reason behind GLP created
Advantages and disadvantages of GLP
Objectives of GLP
Practice of GLP
b pharma 6th sem
pharmaceutical quality assurance
enzyme immobilization, pharmaceutical biotechnology, b pharam 6th sem, PCI.
history,
why immobilise
advantages and disadvantages of enzyme immobilization
methods of enzyme immobilization
physical retention
entrapment
microencapsulation
chemical bonding
adsorption
cross linking
covalent binding
ionic binding
Applications of Immobilized Enzymes
Enzyme utilization in Industry
BIOSENSOR, PHARMACEUTICAL BIOTECHNOLOGY, B PHARAM, 6TH SEM
Basic components of Biosensor
Working of Biosensor
Types of Biosensor
Electrochemical biosensor
Optical biosensor
Thermal biosensor
Resonant biosensor
Ion-sensitive biosensor
Applications of Biosensor
Nano sensors
sensing device
Father of the Biosensor
components of BIOSENSOR
BASIC PRINCIPLE OF BIOSENSOR
BIO-ELEMENT
TRANSDUCER
DETECTOR
RESPONSE FROM BIO-ELEMENT
IDEAL BIOSENSOR
BASIC CHARACTERESTICS
genetic engineering, principles, b pharma 6th sem, biotechnology
What is a gene ?
Definition
History
Process
Molecular tools of genetic engineering
Restriction enzymes
History of restriction enzyme
Mechanism of action
Types of restriction enzymes
Application of restriction enzymes
Blunt ends
Sticky ends
transgenic
cisgenic.
knockout organism.
Host organism vector
TRANSGENIC PLANTS
DOLLY THE SHIP
TRANSGENIC ANIMALS
This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
He discussed the concept of quality improvement, emphasizing its applicability to various aspects of life, including personal, project, and program improvements. He defined quality as doing the right thing at the right time in the right way to achieve the best possible results and discussed the concept of the "gap" between what we know and what we do, and how this gap represents the areas we need to improve. He explained the scientific approach to quality improvement, which involves systematic performance analysis, testing and learning, and implementing change ideas. He also highlighted the importance of client focus and a team approach to quality improvement.
How to Split Bills in the Odoo 17 POS ModuleCeline George
Bills have a main role in point of sale procedure. It will help to track sales, handling payments and giving receipts to customers. Bill splitting also has an important role in POS. For example, If some friends come together for dinner and if they want to divide the bill then it is possible by POS bill splitting. This slide will show how to split bills in odoo 17 POS.
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
Ethnobotany and Ethnopharmacology:
Ethnobotany in herbal drug evaluation,
Impact of Ethnobotany in traditional medicine,
New development in herbals,
Bio-prospecting tools for drug discovery,
Role of Ethnopharmacology in drug evaluation,
Reverse Pharmacology.
The Art Pastor's Guide to Sabbath | Steve ThomasonSteve Thomason
What is the purpose of the Sabbath Law in the Torah. It is interesting to compare how the context of the law shifts from Exodus to Deuteronomy. Who gets to rest, and why?
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
3. BLOTTING
• A molecular technique of transferring proteins, DNA or RNA, onto a
carrier or membrane. Done after a gel electrophoresis, transferring the
molecules from the gel onto the blotting membrane or adding the samples
directly onto the membrane.
4. BLOTTING
TRANSFER PROCESS:
Capillary Method (Wet transfer method): The biomolecules are transferred from the gel
to the membrane using the capillary transfer method. The buffer moves from a high
potential area to low potential area, carrying the biomolecules along with it. This
facilitate movement from the gel to membrane.
Electro-blotting: Relies upon current and a transfer buffer solution to drive proteins or
nucleic acids onto a membrane. In the electrophoretic or electro-blotting transfer
technique, the gel and transfer paper are disposed between two electrodes which serve to
produce an electric field there between. The proteins, nucleic acids, and like specimens
are driven out of the gel and onto the transfer sheet by means of the electric field.
Vacuum blot Method: In a vacuum blot, instead of using capillary forces, the force that will
transfer the DNA is a vacuum. Vacuum blotting can be performed in two hours. It is
therefore a much faster method of transferring DNA. The membrane can then be used in
hybridization experiments.
5. BLOTTING MEMBRANES
• TYPES OF MEMBRANES USED IN SOUTHERN & NORTHERN
BLOTTING:
– Aminobenzyloxymethyl Cellulosic Membranes: Modified cellulosic paper
substituted with m-aminobenzyloxymethyl groups, ideal for blotting of RNA.
•
•
The amino moieties in the groups are activated prior to use by conventional diazotization techniques. The
heterocyclic bases on nucleic acids and nitrogenous heterocyclic bases on proteins form covalent bonds
with the phenyl ring containing the diazo group.
• Most preffered used for Northern Blotting. RNA binding with Nitrocellulose membrane moiety is unstable.
Most stable binding of RNA in amino benzyloxymethyl filter paper.
6. TYPES OF MEMBRANES USED IN WESTERN BLOTTING:
– Nitrocellulose and Supported Nitrocellulose: Mostly used for western blotting
(Protein) and is still a popular membrane for this procedure. Protein binding to
nitrocellulose is instantaneous, nearly irreversible. Nitrocellulose is easily wetted in water
or transfer buffer and is compatible with a wide range of protein detection systems.
Unsupported nitrocellulose is innately fragile and is not recommended for stripping and
reprobing. Supported nitrocellulose is an inert support structure with nitrocellulose
applied to it. The support structure gives the membrane increased strength and resilience.
Supported nitrocellulose can withstand reprobing and autoclaving (121°C) and retains the
ease of wetting and protein binding features of nitrocellulose.
– Polyvinylidene Difluoride (PVDF) Membranes: ideal support for N-terminal
sequencing, amino acid analysis, and immunoassay of blotted proteins (western blotting).
PVDF retains proteins during exposure to acidic or basic conditions and in the presence
of organic solvents. Exhibit better binding efficiency of electroblotted material in the
presence of SDS in the transfer buffer. PVDF membranes must be wetted in 100%
methanol prior to use, but once wet may be used with a transfer buffer that contains no
methanol.
7. Southern Blotting
• This method Involves separation, transfer and hybridization. Technique
developed by E.M. Southern in 1975
• The Southern blot is used to detect the presence of a particular piece of
DNA in a sample.
• The DNA detected can be a single gene, or it can be part of a larger
piece of DNA such as a viral genome.
• 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 DNA
8. Methods in Southern Blotting
• Restriction endonucleases cut
high-molecular-weight DNA
•
strands into smaller fragments.
The mixture of fragmented DNAs
is separated in agarose gel
•
electrophoresis.
The restriction fragments present
in the gel are denatured with
alkali and transferred onto a
nitrocellulose filter or nylon
membrane by blotting.
Fig 1: EcoRI Restriction Endonuclease
Fig 2: Agarose gel electrophoresis
9. Methods in Southern Blotting
• This procedure preserves the
•
distribution of the fragments in
the gel creating a replica of the
gel on the filter.
A sheet of nitrocellulose or nylon
membrane is placed on top of the
gel. Pressure is applied evenly to
the gel to ensure good and even
contact between gel and
membrane.
Fig 4: DNA Denaturation by NaOH
10. Methods in Southern Blotting
• Membrane is then baked in a
vacuum or regular oven at 80 °C
for 2 hours or exposed to
ultraviolet radiation (nylon
membrane) to permanently attach
the transferred DNA to the
•
membrane.
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.
Fig 3: Baking at 80 °C
11. Southern Blotting Applications
•
•
•
•
•
•
•
Identify specific DNA sequences in DNA samples
Isolate desired DNA for construction of rDNA.
Identify mutations, deletions and gene rearrangements.
In GMOs, used for testing to ensure that a particular section of DNA of
known genetic sequence has been successfully incorporated into the
genome of the host organism.
Phylogenetic analysis
To determine the number of copies of a particular DNA sequence
In DNA Fingerprinting for:
– Paternity & Maternity tests
– Forensics
– Personal/ Bio identification
12. 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 in 1979 and was named such by analogy to Southern
blotting.
Northern blot analysis reveals information about RNA identity, size, and
abundance, allowing a deeper understanding of gene expression levels.
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.
13. Northern Blotting Method
• Extraction of total RNA from a
homogenized tissue sample or
from cells.
• mRNA isolated through the use of
oligo (dT) cellulose
chromatography to isolate only
•
•
those RNAs with a poly(A) tail.
RNA samples are then separated
by gel electrophoresis.
Transferred to a nylon membrane
through a capillary or vacuum
blotting system.
14. Northern Blotting Method
• membrane with a
charge is the most
A nylon
positive
effective
blotting
for
since
use in
the
northern
negatively
•
charged nucleic acids have a high
affinity for them.
Once RNA has been transferred
immobilized
to the membrane, it is
through covalent
linkage to the membrane by UV
light or heat. After a probe has
been labeled, it is hybridized to
the RNA on the membrane.
15. Northern Blotting Application
•
•
•
•
Observe a particular gene's expression pattern between tissues, organs,
developmental stages, environmental stress levels, pathogen infection, and
over the course of treatment.
Used to show overexpression of oncogenes and down regulation of tumor-
suppressor genes in cancerous cells.
Detecting a specific mRNA in sample, used for screening recombinants
which are successfully transformed with transgene.
mRNA splicing studies
16. Western Blotting
•
•
•
Detect specific proteins in a sample of tissue homogenate or extract using
labelled antibodies.
The method originated in the laboratory of Harry Towbin at the Friedrich
Miescher Institute, Switzerland in 1979.
The name western blot was given to the technique by W. Neal Burnette and
is a play on the name Southern blot.
17. Western Blotting Method
•
•
Proteins of the sample are separated by gel electrophoresis. Separation of
proteins may be by isoelectric point (pI), molecular weight, electric charge,
or a combination of these factors.
Most common type of gel electrophoresis employs polyacrylamide gels
and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS
polyacrylamide gel electrophoresis) maintains polypeptides in a denatured
state once they have been treated with strong reducing agents to remove
secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl
groups [SH and SH]) and thus allows separation of proteins by their
molecular weight.
18. Western Blotting Method
• Proteins are transferred from the
gel onto a membrane made of
nitrocellulose or polyvinylidene
difluoride (PVDF) for
accessibility to antibodies.
• Transferring by electroblotting
uses an electric current to pull
proteins from the gel into the
PVDF or nitrocellulose
membrane. The proteins move
from within the gel onto the
membrane while maintaining the
organization they had within the
gel.
19. Western Blotting Method
• Protein binding is based upon hydrophobic interactions, as well as charged
interactions between the membrane and protein.
• The uniformity and overall effectiveness of transfer of protein from the gel
to the membrane can be checked by staining the membrane with Coomassie
Brilliant Blue.
• Blocking of non-specific binding to the membrane by dilute solution of
protein -typically 3-5% Bovine serum albumin
• (BSA) or non-fat dry milk Tris-Buffered Saline (TBS) with a minute
percentage (0.1%) of detergent such as Tween 20 or Triton X-100.
20. Western Blotting Method
• The membrane is "probed" for the protein of interest with a modified
antibody which is linked to a reporter enzyme; when exposed to an
appropriate substrate, this enzyme drives a colourimetric reaction and
produces a color.
21. Western Blotting Applications
•
•
•
The confirmatory HIV test employs a western blot to detect anti-HIV
antibody in a human serum sample (ELISA).
Technique for the detection and analysis of proteins.
Diagnostics of various diseases (viral and autoimmune disease included)