Western blotting is a technique used to detect specific proteins in a sample based on antibody-protein binding. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a targeted protein. The process includes denaturing proteins, separating them by SDS-PAGE gel electrophoresis based on size, transferring to a membrane, blocking non-specific binding sites, incubating with primary and secondary antibodies, and detecting the targeted protein.
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.
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.
gives a very brief info about western blotting procedures, attractive slides, with creative animation effects, i hope this ppt of mine works good for seminar and for educational purposes.
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
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
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.
gives a very brief info about western blotting procedures, attractive slides, with creative animation effects, i hope this ppt of mine works good for seminar and for educational purposes.
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
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
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.
This presentation includes the principle involved, chemistry, procedure, and application of various advance molecular biology like SDS PAGE, Western Blotting, and ELISA.
SDS PAGE is widely used to analyze the proteins in complex extracts.
The polyacrylamide gels are used to separate proteins.
Polyacrylamide is inert, and hence, shows no interaction with the protein being separated and forms a matrix.
Size of the pores in the gel can be controlled by adjusting the concentration of acrylamide.
Acrylamide undergoes polymerization in order to form a gel. Hence, APS (ammonium per sulphate) & TEMED (N,N,N’,N’-tetramethylethylenediamine) are added to initiate the process of polymerization.
It's application includes separation of protein mixture on separating gel and their identification using different techniques like western blotting.
Western blotting, also known as immunoblotting or protein blotting, is a core technique in cell and molecular biology. In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells.
Western blots are effective in detecting low nanogram to low picogram amounts of target protein, depending on the antibodies used and the detection substrate chosen. If the target is suspected to be of very low abundance, or if there is no detectible signal on the blot, then it may be necessary to concentrate, immunoprecipitate, or fractionate the starting material.
This technique is used to study cell signalling pathways, cell cycle pathways, drug action pathways, protein-protein interaction.
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones.
In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme.
Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product.
The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
ELISA begins with a coating step, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate.
This is followed by a blocking step in which all unbound sites are coated with a blocking agent.
Following a series of washes, the plate is incubated with enzyme-conjugated antibody.
Another series of washes removes all unbound antibody.
A substrate is then added, producing a calorimetric signal. Finally, the plate is read.
It's types include Direct ELISA, Indirect ELISA, Sandwich ELISA and competitive ELISA. This technique is used to determine serum antibody concentrations, potential food allergens (milk, peanuts, almonds), detection of antigens and antibodies, disease outbreaks.
Introduction and Description to Western Blotting, Steps involved in Western Blotting- Sample Preparation, Protein Gel Electrophoresis, SDS-PAGE, Protein Transfer, Electrophoretic Protein Transfer, Transfer Sandwich Diagram, Blocking, Antibody Probing and Detection, Applications of Western Blotting.
GLP applies to nonclinical studies conducted for the assessment of the safety or efficacy of chemicals (including pharmaceuticals).
GLP helps assure regulatory authorities that the data submitted are a true.
SMGT is a simple and efficient technique to produce transgenic mice
is a transgenic technique, uses the sperms as a natural vector to transport the internalize exogenous DNA into an oocyte during fertilization to produce genetically modified organisms.
Ribozymes (ribonucleic acid enzymes) are RNA molecules that are capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes.
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.
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.
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Palestine last event orientationfvgnh .pptxRaedMohamed3
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http://sandymillin.wordpress.com/iateflwebinar2024
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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.
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.
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The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
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2. Western blotting
• Western blotting is a widely used technique
for the detection and analysis of proteins
based on their ability to bind to specific
antibodies.
• It was first described by Towbin, et.al in 1979.
• Western blotting is a accomplished rapidly,
using simple equipment and inexpensive
reagents, it is commonly used laboratory
technique.
3. Principle
• Western blotting is the transfer of proteins from the SDS-
PAGE gel to a solid supporting membrane.
• Electrophoresis is used to separate complex mixtures of
proteins denaturing discontinuous one dimensional gel
electrophoresis separates proteins only based on molecular
size as they move through a SDS- polyacrylamide gel(SDS
PAGE) toward the anode with the smaller protein migrating
faster and bigger proteins running slower.
• A protein sample is subjected to polyacrylamide gel
electrophoresis. After this the gel is placed over a sheet of
nitrocellulose and the protein in the gel is
electrophoretically transfered to the nitrocellulose.
4. Principle
• The nitrocellulose is then soaked in blocking buffer (3%
skimmed milk solution) to "block" the non-specific
binding of proteins.
• The nitrocellulose is then incubated with the specific
antibody for the protein of interest.
• The nitrocellulose is then incubated with a second
antibody, which is specific for the first antibody.
• The second antibody will typically have a covalently
attached enzyme which, when provided with a
chromogenic substrate, will cause a color reaction
5. Step 1: Sample Prepration
• First step in the sample prepration is isolating
the protein from a sample. Usually proteins
are purified from cells.
6. • Next the protein concentration is determined.
Sample buffer commonly Leammli buffer,
which contains Sodium dodecyl Sulpate (SDS)
and beta- mercaptoethanol (BME) is added to
the protein suspension
7. • The sample buffer is the centrifuge and
heated to near- boiling, which denatures the
protein and allows the SDS to bind to the
protein. SDS carries negative charge. Glycerol
to make samples sink into wells and the Tris
base provides appropriate pH. The blue dye to
visualize samples as gel is run.
8. Step 2: SDS-PAGE
• SDS-PAGE
(sodium dodecylsulphate-polyacrylamide gel electrophoresis)-
method for separation of proteins according to their molecular
weight
• Levels of proteins
• Primary structure = linear chain of amino acids
• Secondary structure = domains of repeating structures, such as β-
pleated sheets and α-helices
• Tertiary structure = 3-dimensional shape of a folded polypeptide,
maintained by disulfide bonds, electrostatic interactions,
hydrophobic effects
• • Quaternary structure = several polypeptide chains associated
together to form a functional protein
9. • proteins denatured by heating them in a
sample buffer containing sodium dodecyl
sulphate (SDS)-the proteins no longer have
any secondary, tertiary or quaternary
structure resultant proteins take on a rod-like
shape and a uniform negative charge-to-mass
ratio proportional to their molecular weights
10. What is in the Sample Buffer?
*Tris buffer to provide appropriate pH
*SDS (sodium dodecyl sulphate) detergent to
dissolve proteins and give them a negative
charge
*Glycerol to make samples sink into wells
*Bromophenol Blue dye to visualize samples
11.
12. Step 3 : Membrane Transfer (Wet
Transfer)
• After the gel is run, it is placed against a membrane , and current is
passed across the gel to the membrane, transferring the proteins
onto the membrane.
• The transferring method is also known as semi- dry method. The
membrane is usually made of PVDF or nitrocellulose, both of which
have advantages and disadvanatages that should be thoroughly
researched prior to use.
13. Step 4: Immunoblotting
• The first step in immunoblotting is to wash the
membrane and block it with non-specific
protein. The non- specific protein binds to the
surface of the membrane where protein is not
already present. This will prevent antibody
from binding to the membrane and giving a
non- specific signal.
•
14. • Next, the primary antibody is added to the
solution in which the membrane is floating.
Remember that the primary antibody
recognizes a specific amino-acid sequence of a
particular protein.
15. • After a wash is conducted to remove unbound primary
antibody, secondary antibody is added. Secondary
antibody recognizes the primary antibody, and usually
is conjugated with an enzyme, such as HRP (Horse
Radish Peroxidase)
• Lastly, another wash is performed to remove unbound
secondary antibody. Non-specific binding of both the
primary and secondary antibodies can occur, but
thorough washing usually minimizes this problem. The
amount of time the primary and secondary antibodies
are applied, directly affects the specificity and strength
of binding.
16. Step 5: Detection
• The detection method used is dependent upon the enzyme to
which the secondary antibody is conjugated. The most common
enzyme used in Western Blotting is HRP, and the substrate used for
detection is known as chemiluminescent substrate.
17. • Once the substrate has been added, the light
being emitted can be detected with film or a
photo imager.