The document discusses SDS gel electrophoresis and blotting techniques. It provides an overview of SDS-PAGE, describing how SDS denatures and coats proteins with negative charges. This allows proteins to be separated by size when run on a polyacrylamide gel with an electric current. It also summarizes Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins. All involve separating molecules, transferring them to a membrane, and using probes or antibodies to identify specific sequences or proteins. The techniques have applications in research, forensics, medicine and molecular biology.
Two-dimensional gel electrophoresis (2-DE) is considered a powerful tool for proteomics work. 2-DE separates proteins depending on two differ steps: the first one is called isoelectric focusing (IEF) which separates proteins according to isoelectric points (pI); the second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which separates proteins based on the molecular weights.
Our website: www.creative-proteomics.com
Two-dimensional gel electrophoresis (2-DE) is considered a powerful tool for proteomics work. 2-DE separates proteins depending on two differ steps: the first one is called isoelectric focusing (IEF) which separates proteins according to isoelectric points (pI); the second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which separates proteins based on the molecular weights.
Our website: www.creative-proteomics.com
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.
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
Standard test that used to determine the charged molecules, mainly proteins and nucleic acids.
Widely used in biochemistry, forensics, genetics and molecular biology.
Laemmli system of SDS-PAGE was first introduced in 1970s
PCR is a revolutionary molecular biology technique used for enzymatically replicating DNA . This technique allows a small amount of DNA molecule to be amplified many times in an exponential manner . It is commonly used in medical and biological research labs for variety of tasks such as detection of hereditary disease , identification of genetic fingerprints diagnosis of infectious disease , cloning of genes and paternity testing .
Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies . The thermostability of DNA polymerases is defined by how long they remain active at the extreme range of temperatures used in PCR.
There have been various thermostable polymerases identified to date, each with its optimal temperature for activity and a unique half-life profile at temperatures greater than 95°C. For example, the half-life of Taq polymerase at 95°C is 40 minutes, whereas the half-life of the hyperthermophilic Deep Vent DNA polymerase extracted from the Pyrococcus species GB-D is several hours at 98–100°C. Polymerase processivity is defined as the number of consecutive nucleotides a single enzyme can incorporate before being dislodged from the DNA template.
At 75°C, native Taq polymerases can typically amplify DNA at a rate of 10–45 nucleotides per second - that’s approximately 2 kilobases per minute!
Some DNA polymerases have been engineered to improve their binding domain, thus making them more stable than conventional Taq. For example, KAPA2G polymerase has a speed of ~150 nucleotides per second - 3-fold higher than Taq. Direct PCR cloning methods include TA and GC cloning, as well as TOPO® Cloning, and enable direct cloning of PCR fragments. For example, the TA cloning approach takes advantage of the 3’ A overhang naturally added to products by Taq polymerase following PCR. The resulting sticky ends then enable recombination with DNA fragments containing 3’ T overhangs, such as linearized vectors.
During indirect PCR cloning, the PCR products are modified prior to recombination with other DNA sequences. For example, in restriction cloning, restriction sites are frequently introduced via PCR to enable restriction digestion and ligation with linearized vectors. PCR mutagenesis is a technique used to generate site-directed sequence changes such as base substitutions, inserts and deletions.
To insert a single point mutation via mutagenesis, for example, PCR primers are designed that contain the desired base change, usually in the middle of the primer sequence. PCR is then performed with the mutagenic primers and a high-fidelity DNA polymerase, which results in the incorporation of the desired mutation into the original sequence.Allele-specific PCR is used to detect sequence variations and ultimately determine the genotype of an organism.
For allele-specific PCR, primers are designed to flank the region of interest. The most common application of PCR is gene expression analysis
2-D electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples.
Two-dimensional electrophoresis was first introduced by O’Farrell and Klose in 1975
2-DGE is a multi-step separation technique in which proteins are solubilized and separated according to charge (pI) in the first dimension using IEF, followed by size (molecular weight, MW) using SDS-PAGE in the second dimension.
The separated proteins are stained with coomassie or silver stain to produce a two-dimensional protein reference map.
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.
This presentation gives an easy introduction to ChIP-seq analyses and is part of a bioinformatics workshop. The accompanying websites are available at http://sschmeier.github.io/bioinf-workshop/#!galaxy-chipseq/
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.
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
Standard test that used to determine the charged molecules, mainly proteins and nucleic acids.
Widely used in biochemistry, forensics, genetics and molecular biology.
Laemmli system of SDS-PAGE was first introduced in 1970s
PCR is a revolutionary molecular biology technique used for enzymatically replicating DNA . This technique allows a small amount of DNA molecule to be amplified many times in an exponential manner . It is commonly used in medical and biological research labs for variety of tasks such as detection of hereditary disease , identification of genetic fingerprints diagnosis of infectious disease , cloning of genes and paternity testing .
Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies . The thermostability of DNA polymerases is defined by how long they remain active at the extreme range of temperatures used in PCR.
There have been various thermostable polymerases identified to date, each with its optimal temperature for activity and a unique half-life profile at temperatures greater than 95°C. For example, the half-life of Taq polymerase at 95°C is 40 minutes, whereas the half-life of the hyperthermophilic Deep Vent DNA polymerase extracted from the Pyrococcus species GB-D is several hours at 98–100°C. Polymerase processivity is defined as the number of consecutive nucleotides a single enzyme can incorporate before being dislodged from the DNA template.
At 75°C, native Taq polymerases can typically amplify DNA at a rate of 10–45 nucleotides per second - that’s approximately 2 kilobases per minute!
Some DNA polymerases have been engineered to improve their binding domain, thus making them more stable than conventional Taq. For example, KAPA2G polymerase has a speed of ~150 nucleotides per second - 3-fold higher than Taq. Direct PCR cloning methods include TA and GC cloning, as well as TOPO® Cloning, and enable direct cloning of PCR fragments. For example, the TA cloning approach takes advantage of the 3’ A overhang naturally added to products by Taq polymerase following PCR. The resulting sticky ends then enable recombination with DNA fragments containing 3’ T overhangs, such as linearized vectors.
During indirect PCR cloning, the PCR products are modified prior to recombination with other DNA sequences. For example, in restriction cloning, restriction sites are frequently introduced via PCR to enable restriction digestion and ligation with linearized vectors. PCR mutagenesis is a technique used to generate site-directed sequence changes such as base substitutions, inserts and deletions.
To insert a single point mutation via mutagenesis, for example, PCR primers are designed that contain the desired base change, usually in the middle of the primer sequence. PCR is then performed with the mutagenic primers and a high-fidelity DNA polymerase, which results in the incorporation of the desired mutation into the original sequence.Allele-specific PCR is used to detect sequence variations and ultimately determine the genotype of an organism.
For allele-specific PCR, primers are designed to flank the region of interest. The most common application of PCR is gene expression analysis
2-D electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples.
Two-dimensional electrophoresis was first introduced by O’Farrell and Klose in 1975
2-DGE is a multi-step separation technique in which proteins are solubilized and separated according to charge (pI) in the first dimension using IEF, followed by size (molecular weight, MW) using SDS-PAGE in the second dimension.
The separated proteins are stained with coomassie or silver stain to produce a two-dimensional protein reference map.
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.
This presentation gives an easy introduction to ChIP-seq analyses and is part of a bioinformatics workshop. The accompanying websites are available at http://sschmeier.github.io/bioinf-workshop/#!galaxy-chipseq/
Agarose gel electrophoresis by KK Sahu sirKAUSHAL SAHU
INTRODUCTION.
HISTORY.
PROCESS OF GEL ELECTROPHORESIS.
AGAROSE GEL ELECTROFORESIS.
POLYACRYALAMIDE GEL ELECTRIPHORESIS.
GEL CONDITION.
DENATURETION.
NATIVE.
BUFFERS.
USES.
CONCLUSION.
REFFERENCES.
This presentation consist of all information regarding sodium dodecyl sulphate PAGE. The general information regarding electrophoresis and its main types are also included in this presentation.
Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge.
Different types of electrophoresis.
Gel electrophoresis; Agarose Gel electrophoresis; polyacrylamide gel electrophoresis; pulsed-field gel electrophoresis
electrophoresis: types, advantages, disadvantages and applications.Cherry
Electrophoresis is a general term that describes the migration and separation of charged particles under the influence of an electric field.
The particles maybe simple ions, complex macromolecules and colloids or particulate matter- either living cells such as bacteria or inert material such as oil emulsion, droplet etc.
The pores present in the gel work like a sieve, allowing the smaller molecules to pass through more quickly and easily than the larger molecules.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
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Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
NVBDCP.pptx Nation vector borne disease control program
Gel electroporosis
1. SDS GEL ELECTROPHORESIS
AND BLOTTING TECHNIQUES.
By
Y.PRATHAP
M.Pharm I- Sem (PHARMACEUTICS).
Roll no: 256213886031.
Under the guidance
of:
Asst.Prof . Mr. Uttam
Prasad Panigrahi .
M.PHARM,(PAT).
3. INTRODUCTION OF SDS-PAGE (POLYACRYLAMIDE
GEL ELECTROPHORESIS)
SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis, is a
technique widely used in biochemistry,
forensics, genetics and molecular biology:
to separate proteins according to their
electrophoretic mobility (a function of length of
polypeptide chain or molecular weight).
to separate proteins according to their size, and
no other physical feature.
4. …SDS-PAGE
SDS (sodium dodecyl sulfate) is a detergent
(soap) that can dissolve hydrophobic molecules
but also has a negative charge (sulfATE)
attached to it.
5. Fig.1Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing
negative and positive charges due to the charged R-groups in the protein.
The large H's represent hydrophobic domains where nonpolar R-groups have collected in an
attempt to get away from the polar water that surrounds the protein.
After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative
charges which overwhelms any positive charges the protein had due to positively charged R-groups.
The resulting protein has been denatured by SDS (reduced to its primary structure-amino
acid sequence) and as a result has been linearized.
6. ..SDS
SDS (the detergent soap) breaks up hydrophobic
areas and coats proteins with negative charges
thus overwhelming positive charges in the
protein.
The detergent binds to hydrophobic regions in a
constant ratio of about 1.4 g of SDS per gram of
protein.
• Therefore, if a cell is incubated with SDS, the
membranes will be dissolved, all the proteins
will be solubalized by the detergent and all the
proteins will be covered with many negative
charges.
7. PAGE
If the proteins are denatured and put into an
electric field (only), they will all move
towards the positive pole at the same rate,
with no separation by size.
However, if the proteins are put into an
environment that will allow different sized
proteins to move at different rates.
The environment is polyacrylamide.
the entire process is called polyacrylamide
gel electrophoresis (PAGE).
8. ..PAGE
Small molecules move through the polyacrylamide
gel faster than big molecules.
Big molecules stays near the well.
9. PRINCIPLE:
PAGE (Polyacrylamide Gel Electrophoresis), is an
analytical method used to separate components of a
protein mixture based on their size. The technique is
based upon the principle that a charged molecule will
migrate in an electric field towards an electrode with
opposite sign.
The proteins being covered by SDS are negatively
charged and when loaded onto a gel and placed in an
electric field, it will migrate towards the anode
(positively charged electrode) are separated by a
molecular sieving effect based on size. After the
visualization by a staining (protein-specific) technique.
11. APPARATUS:
Apparatus for gel electrophoresis are relatively simple .
Electrophoresis cells are essentially plastic boxes with anode and
cathode compartments. Electrodes(usually platinum wire) and jacks
for making electrical contact with the electrodes.
Gels are held vertically between the electrode chambers during the
run. Gel cassettes have open tops and bottoms. The bottom is sealed
with a gasket during gel formation and the top is open to receive
monomer solution. The top and bottom ends are open and in contact
with buffer for electrophoresis.
High voltage direct current supplies provide electrical power for
electrophoresis.
Micropipettes, test tubes and heating blocks are the sample handling
necessities.
12. PARTS OF THE SYSTEM
Gel support medium
Agarose.
Polyacrylamide(PA).
Detergent: sodium dodecyl sulfate(SDS).
Buffer : the electrical current in an electrophoresis cell is
carried largely by the ions supplied by the buffer compounds.
Proteins constitute only a small portion of the current carrying
ions in an electrophoresis cell. So buffers supply current
carrying ions, maintain desired PH, provide a medium for heat
for dissipation . Ex : Tris-acetate-EDTA and Tris- borate -
EDTA.
DC Power supply.
13.
14.
15. PROCESS:
Sample preparation.
Prepare 2X sample buffer consisting of 0.5MTris-HCl,
pH 6.8, 4.4% SDS, 300mMMercaptoethanol,
10mg/ml Bromophenol Blue and mix with equal volume
of sample .Bring to 95° C for 10 minutes, cool to room
temperature before loading. If particulate is present,
centrifuge samples 5 minutes at 14k RPM in
microcentrifuge, and load the gel.
17. PROTEIN VISUALIZATION ON GELS
• Immediately after electrophoresis proteins in the gels are
precipitated by either adding alcohol containing
solutions or strong acids (e.g. TCA)
• DNA may be visualized using ethidium bromide.
Protein are often stained by Coomassie Brilliant Blue
dye or by photography-like treatment with AgNO3
(silver staining)
There are many other stains available (e.g. Stains-all,
fluorescence probes etc.)
20. APPLICATIONS:
SDS PAGE is a useful method for separating and
characterizing macromolecules like DNA, RNA and
proteins.
In Forensic , DNA Fingerprinting: men proving or
disproving paternity by this technique. Used as witness.
The human Genome project.
Illness: It can help scientists to identify certain damaged
genes . It can also help to identify certain genetic
diseases like sickle cell anemia, also identify viruses.
Blotting : Separation of restricted genomic DNA prior to
southern blotting and RNA prior to northern blotting.
22. WHAT IS BLOTTING?
Technique for transferring
DNA
RNA
Proteins
onto a carrier so they can be separated,
and often follows the use of a gel
electrophoresis.
23. TYPES OF BLOTTING TECHNIQUES
BLOTTING
TECHNIQUES
Southern Blot
It is used to detect
the DNA.
Northern blot
It is used to detect
the RNA.
Western blot
It is used to detect
proteins.
24. BLOTTING SHEET
Whatman 3mm paper….. world’s most widely used
blotting paper.
REASON????
high quality
purity
consistency
25. CREATING THE SANDWICH
The sandwich consists of :
filter paper
Nitrocellulose membrane
gel matrix
another piece of filter paper
27. SOUTHERN BLOTTING
“Used to detect the DNA”
This method Involves:
Separation
Transfer
Hybridization.
This DNA can be:
Single gene
Part of a larger piece of DNA……..viral genome
“The key to this method is Hybridization”
28. HYBRIDIZATION
“Process of forming a
dsDNA molecule between a
ssDNA probe and a ss-target
patient DNA”
29. PRINCIPLE
The mixture of molecules is separated.
Immobilized on a matrix.
Probe addition to the matrix to bind to the molecules.
Unbound probes are removed.
“The place where the probe is connected
corresponds to the location of the immobilized
target molecule.”
30. STEPS IN SOUTHERN BLOTTING
The DNA is digested
Fragments
Gel electrophoresis
Transfer to membrane
Probing
Autoradiogram
31.
32. APPLICATIONS
Southern blotting is used in:
Gene discovery
Mapping
Evolution
Development studies
Diagnostics
Forensics
33. NORTHERN BLOTTING
History:
Northern blotting was developed by James
Alwine and George Stark at Stanford University.
Northern blotting is a technique for
detection of specific RNA sequences
34. STEPS INVOLVED IN N.B
RNA isolation
Loading of sample on Agarose gel
Blotting on nitrocellulose membrane
Labeling with probe
Washing to remove unbound probe
Detection by autoradiogram
35.
36. APPLICATIONS
A standard for the direct study of gene expression at the
level of mRNA (mRNA transcripts)
Detection of mRNA transcript size
Study RNA degradation
Study RNA splicing - can detect alternatively spliced
transcripts
Study RNA half-life
Study IRES (internal ribosomal entry site) – to remove
possibility of RNA digestion vs. 2nd cistron translation.
37. WESTERN BLOTTING
Discovery???
Dr. Douglas Lake of the University of Arizona School of
Medicine's Department of Microbiology and Immunology
“A technique in which proteins are
separated by gel electrophoresis and
transferred to a membrane sheet. A
specific protein is then identified through
its reaction with a labeled antibody.”
39. PREREQUISITE FORW.B
•The SDS
PAGE technique is a
prerequisite for Western
blotting.
“SDS (sodium dodecyl sulfate) is a
detergent (soap) that can dissolve
hydrophobic molecules but also has
a negative charge (sulfate) attached
to it.”
40. STEPS INW.B
1. Gel electrophoresis:
The proteins are separated according to size.
2. Membrane Transfer:
Transferring to nitrocellulose by applying
current.
3. Blocking:
Done to prevent non-specific protein interactions
between the membrane and the antibody protein.
41.
42. APPLICATIONS
To identify the specific proteins
To identify their masses
The confirmatory HIV test to detect anti-HIV antibody in a human
serum sample.
The definitive test for Bovine spongiform encephalopathy (BSE,
commonly referred to as 'mad cow disease').
Some forms of Lyme disease testing also employ Western blotting.
43. REFERENCES:
Introduction to Biotechnology by W.J. Thieman and
M.A. Palladino. Pearson & Benjamin Cummings
2nd edition.
http://www.toodoc.com/SDS-PAGE-ppt.html
http://www.bio.davidson.edu/courses/genomics/met
hod/Westernblot.html
http://en.wikipedia.org/wiki/Dot_blot
Bio analytical techniques by M.L.Srivastava
http://amrita.vlab.co.in/index.php.
Wikipedia.
www.authorstream.com.