This document summarizes the technique of immunoprecipitation. It describes immunoprecipitation as a method to precipitate protein antigens out of solution using an immobilized specific antibody. The document outlines different applications of immunoprecipitation including measuring protein molecular weight, detecting post-translational modifications, and analyzing protein-protein interactions. It also describes different types of immunoprecipitation techniques such as co-immunoprecipitation, chromatin immunoprecipitation, and RNA immunoprecipitation.
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.
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.
Immunoprecipitation: Procedure, Analysis and Applicationsajithnandanam
Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.
Incubate sample with antibody against protein of interest.
Separate antibody-protein complex from remaining sample
Analysis
it will help you to understand how the protein microarrays are made, what are the different types and what all purposes they are used for. its very useful ppt
Immunohistochemistry (IHC) is the localization of a known antigen in tissues by utilizing antibodies directed towards that (specific) antigen. In this presentation, we will introduce the procedure of IHC and the troubleshooting solutions.
Western blot is a commonly used method for protein analysis. It can be used for qualitative and semi-quantitative protein analysis. For the accomplishment of the western blot, there are three elements, separation of proteins by size, transferring proteins to a solid support, and marking proteins by primary and secondary antibodies for visualization.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
(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.
2. Immunoprecipitation
2
• Technique of precipitating protein antigens out of a
solution using a specific antibody which is
immobilized to a solid support.
• Widely used method for protein isolation from
complex samples such as cell lysates, serum and
tissue homogenates.
• Applications:
-to measure the molecular weight of the given
protein
-to determine post translational modifications
-to analyze the expression level of a protein of
interest and
-in the study of protein-protein as well as protein-
nucleic acid interactions.
3. Methods
3
Direct method/Pre-immobilized
antibody approach
-The preferred method when the
target protein is abundant.
-Requires less primary Ab.
Indirect method/Free antibody
approach
-The preferred method when the
Ab has poor affinity for the target or
when the target protein is in low
abundance
4. Magnetic beads vs. agarose resin for
immunoprecipitation
4
Magnetic beads provide the
best balance of capacity/yield,
reproducibility, purity, and cost
savings for routine small-scale
isolation of specific proteins
and protein complexes.
Agarose is most appropriate
for large-scale IP reactions
when sufficient antibody is
plentiful at a low cost and
where the goal is to purify a
sufficient amount of target
protein for multiple
downstream assays.
5. Types of immunoprecipitation
5
Co-immunoprecipitation
Used to analyze protein–protein interactions.
Main purpose of Co-IP is the identification of
interaction partners (other proteins, ligands, co-
factors, or signaling molecules) to the protein of
interest.
6. 6
Chromatin Immunoprecipitation (ChIP)
Used to investigate regions of the genome
associated with a target DNA-binding protein,
or to identify specific proteins associated with
a particular region of the genome.
Main steps: Crosslinking, cell lysis, chromatin
preparation, IP, reverse crosslinking, DNA
purification and quantitation.
7. 7
RNA Immunoprecipitation (RIP)
• Targets RNA-binding proteins (ribonucleoproteins).
• Performed using an antibody that targets a specific
RNA-binding protein.
• RNA-protein complexes are separated by RNA
extraction.
8. 8
Tagged protein IP
Proteins can be tagged with an epitope to which a high affinity
antibody is available and expressed in the cell of interest.
Tags can be either short peptide sequences or fluorescent proteins,
including: FLAG, c-Myc, Hemagglutinin (HA), Green fluorescent
protein (GFP).
Limitation:
Overexpressed tagged protein is immunoprecipitated
Tagging the protein may interfere with protein function.
10. IP Buffers
10
Lysis buffers
Stabilizes native protein conformation,
Inhibits enzymatic activity,
Maximizes the release of proteins from the cells or tissue.
Non-denaturing buffers: NP-40 or Triton X-100
Denaturing buffers: radio-immunoprecipitation assay (RIPA)
Contains NaCl and Tris-HCl and have a slightly basic pH (7.4 to 8).
Proteasomal inhibitors: PMSF, aprotinin and leupeptin
11. 11
Wash buffers
Multiple washes with simple wash buffers, such as PBS either alone
or with low detergent concentrations can be used to remove
contaminants.
Elution buffers
Elution directly in reducing SDS-PAGE sample buffer.
Non-denaturing elution buffer for protein purification: 0.1 M glycine at
pH 2.5 to 3.
Low pH condition dissociates antibody-antigen interactions, as well
as the antibody-Protein A/G interaction.
