Pharmaceutical biotechnology ..in that different blotting techniques such as ELISA , western blotting and southern blotting.There applications and their advantage and disadvantage with their diagrams
Pharmaceutical biotechnology ..in that different blotting techniques such as ELISA , western blotting and southern blotting.There applications and their advantage and disadvantage with their diagrams
PRINCIPLE & APPLICATIONS OF IMMUNO BLOTTING TECHNIQUES.pptxAnupkumar Sharma
The presentation focuses on the ELISA and immunoblotting techniques like southern blotting, western blotting etc.
It describes principle, method, advantages and disadvantages and applications of different types of ELISA, steps involves and applications of western blotting and southern blotting techniques.
Since antigen and antibody reactions are specific, they can be used to identify each other.
These diagnostic tests are particularly useful in diagnosing for examples: infectious diseases, autoimmune diseases, and in typing of blood and tissues prior to transplantation.
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.
OTECHNOLOGY IS CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ALSO .....THEIR INTERESTING PART IS TO LEARN ABOUT MICROBIAL GENETICS AND THEIR METHODS OF GENE TRANSFER
PRINCIPLE & APPLICATIONS OF IMMUNO BLOTTING TECHNIQUES.pptxAnupkumar Sharma
The presentation focuses on the ELISA and immunoblotting techniques like southern blotting, western blotting etc.
It describes principle, method, advantages and disadvantages and applications of different types of ELISA, steps involves and applications of western blotting and southern blotting techniques.
Since antigen and antibody reactions are specific, they can be used to identify each other.
These diagnostic tests are particularly useful in diagnosing for examples: infectious diseases, autoimmune diseases, and in typing of blood and tissues prior to transplantation.
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.
OTECHNOLOGY IS CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ALSO .....THEIR INTERESTING PART IS TO LEARN ABOUT MICROBIAL GENETICS AND THEIR METHODS OF GENE TRANSFER
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
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.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
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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.
2. • The process of transferring macromolecules from gel to a
membrane followed by their detection on the membrane is known
as Blotting
• DNA – southern blotting
RNA – northern blotting
Proteins – western blotting
• Western blotting allows investigators to determine molecular weight
of protein and to measure relative concentration of protein present
in different samples.
3. • Western blotting technique rely on the specificity of binding
between a molecule of interest and a probe to allow detection of
molecules of interest in a mixture of similar molecules .
• It is used for the identification of particular protein from the
mixture of protein .
• In this method labeled antibody against particular protein is used
to identify desired protein , so it is a specific test .
5. • Tissue preparation
• Gel electrophoresis
• Transfer
• Blocking
• Antibody probing
• Detection
• Analysis
6. TISSUE PREPARATION
• Protein is extracted from cell by mechanical or chemical lysis
• To prevent the digestion of the sample by its own enzyme –anti protease
and phosphatase are used
GEL ELECTROPHORESIS
• Separation of proteins
• SDS-PAGE allows separation of proteins according to molecular weight
• Sample to be analyzed is mixed with SDS.
• SDS is an anionic detergent , imparts negative charge to proteins.
• Polyacrylamide gel
• Buffer used -TRIS
7. TRANSFER
• Following gel electrophoresis, the separated proteins are transferred
from polyacrylamide gel to the membrane.
• Membrane : nitrocellulose or polyvinylidenedifluoride (PVDF)
• The membrane is placed on top of the gel , sandwiched in transfer stack
therefore when protein binds to these membranes , their epitopes are
easily accessible to antibodies.
• Methods : capillary
electrophoretic
• Proteins in the gel are negatively charged ,they move from the gel to the
membrane and becomes attached to it .
BLOCKING
• To prevent non-specific binding of antibodies .
• Antibodies are also proteins so they are likely to bind to nitrocellulose .
• It is essential to block spaces not occupied by proteins.
• Blocking agents used : bovine serum albumin (BSA) or casein
8. ANTIBODY PROBING
The specificity of antigen-antibody interaction permits the
identification of proteins in the sample
1. Primary
• Membrane is incubated with primary antibody .
• The antibody is specific to target protein .
• It binds to the target proteins on the membrane forming
antigen: antibody complex and the excess is washed off .
2. Secondary
• After washing ,the membrane is incubated with secondary
antibody to maximize sensitivity of detection .
• Binding of multiple secondary antibodies to the primary
antibody leads to amplification of detection signals.
• The excess secondary antibody is washed off.
9. DETECTION
• A substrate reacts with the enzyme that is bound to secondary
antibody to generate a coloured band .
• Enzyme – alkaline phosphatase or horseradish peroxidase(HRP)
it generates chemiluminescent signals upon addition of chromogenic
agent .
• The enzyme in the membrane catalysis oxidation of substrate into
an insoluble purple product.
10.
11. • SEPARATION
Proteins are separated by gel electrophoresis, usually sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE).
• TRANSFER
The proteins are transferred to a sheet of special blotting paper called
nitrocellulose or polyvinylidenedifluoride (PVDF).
• PROBING
The blot is incubated with antibody to bind to any remaining sticky places on
nitrocellulose .Secondary antibody is then added to the solution which is
able to bind to its specific protein .The antibody has an enzyme ( e.g. alkaline
phosphatase or horseradish peroxidase ) or dye attached to it .
• DETECTION
The location of antibody is revealed by incubating it with a colorless
substrate that the attached enzyme converts to colored product which can
be seen and photographed.
12. - To determine size and amount of protein in given sample.
- Disease diagnosis : detects antibody against virus or bacteria in
serum .
- Western blotting technique is the confirmatory test for HIV.
It detects anti HIV antibody in patient's serum.
- Useful to detect defective proteins . (prions disease)
- Definitive test for Creutzfeldt-Jakob disease , Lyme disease,
Hepatitis B.
13. Western blot for HIV:-
• Western blot is the highly specific confirmatory test for diagnosis
of HIV .
• The basic technique for WB involves sorting of proteins on gel
followed by its transfer and then probing with antibodies which
react with proteins that are being searched for.
For HIV western blot ,protein (antigen) is known ,they
look for antibodies in patients blood that stick to them.
• Antibodies against the following HIV proteins-
envelope: gp41 and gp 120
core : p17,p24, p55
• Proteins from known HIV –infected cells are separated and blotted
on membrane ,the serum to be tested is applied in the primary
antibody incubation step ,after washing the excess secondary
antibody conjugated with an enzyme is added. The stained band
will indicate whether the patient ‘s serum contains anti-HIV
antibody.
14. IMMUNOLOGY AND IMMUNOTECHNOLOGY
- ASHIM K. CHAKRAVARTY
IMMUNOLOGY - Dr. P MADHAVEE LATHA
https://www.onlinebiologynotes.com/western-blotting-
technique-principle-procedure-application/
https://novowb.wordpress.com/category/western-blot-
applications/
https://www.sigmaaldrich.com/technical-
documents/articles/biology/western-blotting-introduction.html
https://www.bosterbio.com/protocol-and-troubleshooting/western-blot-
principle