Protein-protein interactions are important for many biological processes. There are various types of interactions depending on their composition and duration. Methods to study interactions include yeast two-hybrid, co-immunoprecipitation, affinity chromatography, and chromatin immunoprecipitation. Databases such as IntAct and MINT provide repositories for protein interaction data.
Scoring system is a set of values for qualifying the set of one residue being substituted by another in an alignment.
It is also known as substitution matrix.
Scoring matrix of nucleotide is relatively simple.
A positive value or a high score is given for a match & negative value or a low score is given for a mismatch.
Scoring matrices for amino acids are more complicated because scoring has to reflect the physicochemical properties of amino acid residues.
Ab Initio Protein Structure Prediction is a method to determine the tertiary structure of protein in the absence of experimentally solved structure of a similar/homologous protein. This method builds protein structure guided by energy function.
I had prepared this presentation for an internal project during my masters degree course.
Prediction of the three dimensional structure of a given protein sequence i.e. target protein from the amino acid sequence of a homologous (template) protein for which an X-ray or NMR structure is available based on an alignment to one or more known protein structures
Scoring system is a set of values for qualifying the set of one residue being substituted by another in an alignment.
It is also known as substitution matrix.
Scoring matrix of nucleotide is relatively simple.
A positive value or a high score is given for a match & negative value or a low score is given for a mismatch.
Scoring matrices for amino acids are more complicated because scoring has to reflect the physicochemical properties of amino acid residues.
Ab Initio Protein Structure Prediction is a method to determine the tertiary structure of protein in the absence of experimentally solved structure of a similar/homologous protein. This method builds protein structure guided by energy function.
I had prepared this presentation for an internal project during my masters degree course.
Prediction of the three dimensional structure of a given protein sequence i.e. target protein from the amino acid sequence of a homologous (template) protein for which an X-ray or NMR structure is available based on an alignment to one or more known protein structures
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
The Protein Data Bank (PDB) is a database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. This presentation deals with what, why, how, where and who of PDB. In this presentation we have also included briefing about various file formats available in PDB with emphasis on PDB file format
protein structure prediction methods. homology modelling, fold recognition, threading, ab initio methods. in short and easy form slides. after one time read you can easily understand methods for protein structure prediction.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
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.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
The Protein Data Bank (PDB) is a database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. This presentation deals with what, why, how, where and who of PDB. In this presentation we have also included briefing about various file formats available in PDB with emphasis on PDB file format
protein structure prediction methods. homology modelling, fold recognition, threading, ab initio methods. in short and easy form slides. after one time read you can easily understand methods for protein structure prediction.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
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.
Introduction.
Types of Protein – Protein Interaction.
Methods to investigate Protein – Protein Interaction.
Protein – Protein Interaction modulated by Chemical energy.
Two Hybrid Screening.
Overview of Protein – Protein Interaction analysis.
Biological effect of Protein – Protein interaction.
Conclusion.
Reference.
A genome is an organism’s complete set of DNA or complete genetic makeup, The entire DNA complement. It describes the identity and the sequence of genes of an organism.
Genomics is the study of entire genomes(structure, function, evolution, mapping, and editing of genomes)
Executing the sequencing and analysis of entire human genome enables more rapid and effective identification of disease associated genes and provide drug companies with pre validated targets.
Proteomics is the systematic high-throughput separation and characterization of proteins within biological systems./ large scale study of protein and their functions.
Proteomics measures protein expression directly, not via gene expression, thus achieving better accuracy. Current work uses 2-dimensional polyacrylamide gel electrophoresis(2D- PAGE) and mass spectrometry.
New separation and characterization technologies, such as protein microarray and high throughput chromatography are being developed.
Yeast two-hybrid is based on the reconstitution of a functional transcription factor (TF) when two proteins or polypeptides of interest interact. Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene.
Introduction to proteomics, techniques to study proteomics such as protein electrophoresis, chromatography and mass spectrometry and protein database analysis, case studies derived from scientific literature including comparisons between healthy and diseased tissues, new approaches to analyse metabolic pathways, comprehensive analysis of protein-protein interactions in different cell types.
protein microarray_k.b institute (m.pharm pharmacology) .pptxNittalVekaria
1: Introduction
Welcome to our presentation on Protein Microarrays.
Discover the revolutionary technology transforming protein analysis and biomolecular research
2: What are Protein Microarrays?
Protein microarrays are high-throughput platforms for studying protein-protein interactions, protein function, and biomarker discovery.
They consist of thousands of immobilized proteins on a solid surface, allowing for simultaneous analysis of multiple proteins.
3Components of Protein Microarrays
Substrate: Glass slides, membranes, or beads.
Proteins: Target proteins immobilized on the substrate.
Detection System: Fluorescent dyes, antibodies, or other probes.
Imaging System: Scanners or cameras for data acquisition.
4: Types of Protein Microarrays
Analytical Microarrays: Used for studying protein-protein interactions, protein expression profiling, and protein function analysis.
Antibody Microarrays: Utilized for detecting and quantifying specific proteins or antibodies in biological samples.
Reverse-Phase Protein Arrays (RPPAs): Designed for high-throughput protein expression profiling and signaling pathway analysis.
5:Applications of Protein Microarrays
Biomarker Discovery: Identification of disease-specific biomarkers for diagnosis, prognosis, and treatment monitoring.
Drug Discovery: High-throughput screening of drug candidates and target validation.
Functional Proteomics: Mapping protein-protein interactions, post-translational modifications, and protein function analysis.
Clinical Diagnostics: Detection of infectious diseases, cancer biomarkers, and autoimmune disorders.
6: Workflow of Protein Microarray Experiment
Protein immobilization: Spotting or printing target proteins onto the microarray substrate.
Sample incubation: Incubating the microarray with biological samples containing proteins of interest.
Detection and analysis: Using fluorescent probes or antibodies to detect bound proteins and quantifying the signals.
Data interpretation: Analyzing and interpreting the results to extract meaningful biological insights.
7: Advantages of Protein Microarrays
-High-throughput analysis of thousands of proteins in parallel.
Small sample volume requirement.
Enables multiplexed assays for comprehensive protein profiling.
Facilitates rapid biomarker discovery and validation.
8: Challenges and Considerations
Standardization of protocols and reagents.
Optimization of protein immobilization and detection methods.
Data analysis and interpretation complexities.
Cost and accessibility of microarray platforms.
9: Future Perspectives
Integration with other omics technologies for holistic biological insights.
Development of miniaturized and portable microarray platforms for point-of-care diagnostics.
Advancements in data analysis algorithms and bioinformatics tools.
Expanding applications in personalized medicine and precision healthcare
10: Conclusion
Protein microarrays offer a powerful and versatile tool for protein analysis and biomarker discover
protein microarray_k.b institute (m.pharm pharmacology) .pptxNittalVekaria
1: Introduction
Welcome to our presentation on Protein Microarrays.
Discover the revolutionary technology transforming protein analysis and biomolecular research
2: What are Protein Microarrays?
Protein microarrays are high-throughput platforms for studying protein-protein interactions, protein function, and biomarker discovery.
They consist of thousands of immobilized proteins on a solid surface, allowing for simultaneous analysis of multiple proteins.
3Components of Protein Microarrays
Substrate: Glass slides, membranes, or beads.
Proteins: Target proteins immobilized on the substrate.
Detection System: Fluorescent dyes, antibodies, or other probes.
Imaging System: Scanners or cameras for data acquisition.
4: Types of Protein Microarrays
Analytical Microarrays: Used for studying protein-protein interactions, protein expression profiling, and protein function analysis.
Antibody Microarrays: Utilized for detecting and quantifying specific proteins or antibodies in biological samples.
Reverse-Phase Protein Arrays (RPPAs): Designed for high-throughput protein expression profiling and signaling pathway analysis.
5:Applications of Protein Microarrays
Biomarker Discovery: Identification of disease-specific biomarkers for diagnosis, prognosis, and treatment monitoring.
Drug Discovery: High-throughput screening of drug candidates and target validation.
Functional Proteomics: Mapping protein-protein interactions, post-translational modifications, and protein function analysis.
Clinical Diagnostics: Detection of infectious diseases, cancer biomarkers, and autoimmune disorders.
6: Workflow of Protein Microarray Experiment
Protein immobilization: Spotting or printing target proteins onto the microarray substrate.
Sample incubation: Incubating the microarray with biological samples containing proteins of interest.
Detection and analysis: Using fluorescent probes or antibodies to detect bound proteins and quantifying the signals.
Data interpretation: Analyzing and interpreting the results to extract meaningful biological insights.
7: Advantages of Protein Microarrays
-High-throughput analysis of thousands of proteins in parallel.
Small sample volume requirement.
Enables multiplexed assays for comprehensive protein profiling.
Facilitates rapid biomarker discovery and validation.
8: Challenges and Considerations
Standardization of protocols and reagents.
Optimization of protein immobilization and detection methods.
Data analysis and interpretation complexities.
Cost and accessibility of microarray platforms.
9: Future Perspectives
Integration with other omics technologies for holistic biological insights.
Development of miniaturized and portable microarray platforms for point-of-care diagnostics.
Advancements in data analysis algorithms and bioinformatics tools.
Expanding applications in personalized medicine and precision healthcare
10: Conclusion
Protein microarrays offer a powerful and versatile tool for protein analysis and biomarker discover
role of proteomics in target discovery and validation
1 target of drug action
2 proteomics
3 facts about proteins
4 post translational modification
5 additional modification
6 methods of studying proteins
7 hybrid technologies
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
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.
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.
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.
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.
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.
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.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
2. • Protein–protein interactions occur when two or more proteins bind
together.
• Proteins control and mediate many of the biological activities of cells by
these interactions.
• Information about PPIs improves our understanding of diseases and
can provide the basis for new therapeutic approaches.
• The structure of a protein influences its function by determining the
other molecules with which it can interact and the consequences of those
interactions.
• Multi subunit protein
– hemoglobin, core RNA polymerase, small nuclear
ribonucleoproteins and the ribosome etc..
3. Transient protein interaction: strong and irreversible, it readily undergoes
changes in the oligomeric state.
• Interactions of protein kinases, protein phosphatases, glycosyl
transferases, acyl transferases, proteases, etc., with their substrate
proteins.
– Proteins for Cell growth, cell cycle, metabolic pathways, and
signal transduction
• These interactions are very important in our lives as any disorder in
them can lead to fatal diseases such as Alzheimer’s and Creutzfeld-
Jacob Disease.
• Perhaps the most well known example of Protein-Protein Interaction
is between Actin and Myosin while regulating Muscular contraction
in our body.
4. Types of Protein-Protein Interactions
On the basis of their Composition
Homo-Oligomers: These are macromolecule complexes having one
type of protein subunits.
e.g. : PPIs in Muscle Contraction
Hetero-Oligomers: These are macromolecule complexes having
multiple types protein subunits.
e.g. : PPI between Cytochrome Oxidase and TRPC3 (Transient
receptor potential cation channels
On the basis of duration
• Stable Interactions: These comprise of interactions that last for a
long duration. These Interactions carry out Functional or Structural
roles.
e.g.: Haemoglobin structure
• Transient Interactions : Interactions that last a short period of time.
e.g.: Muscle Contraction
5. Effects of protein interaction
• They can alter the kinetic properties of proteins
• Protein interactions are one common mechanism to allow for
substrate channeling.
• Can result in the formation of a new binding site
• Can inactivate a protein
• Can change the specificity of a protein for its substrate
• Identify the different interactions, understand the extent to which
they take place in the cell, and determine the consequences of the
interaction
7. Another way of classification for methods for identification of PPIs
• The first is ‘atomic observation’ in which the protein interaction
detected using, for example, X-ray crystallography. These
experiments can yield specific information on the atoms or residues
involved in the interaction.
• The second is a ‘direct interaction observation’ where protein
interaction between two partners can be detected as in a two-hybrid
experiment.
• At a third level of observation, multi-protein complexes can be
detected using methods such as immuno-precipitation or mass-
specific analysis. This type of experiment does not reveal the
chemical detail of the interactions or even reveal which proteins are
in direct contact but gives information as to which proteins are found
in a complex at a given time.
8. • This is method that uses transcriptional activity as a measure of
protein-protein interaction
• It relies on the modular nature of many site-specific transcriptional
activators, which consist of a DNA-binding domain and a
transcriptional activation domain
• DNA-binding domain serves to target the activator to the specific
genes that will be expressed
• Activation domain contacts other proteins of the transcriptional
machinery to enable transcription to occur
YEAST TWO HYBRID SYSTEM
9. • yeast two-hybrid (Y2H) system has variations involving different
reagents and has been adapted to high-throughput screening.
• The strategy interrogates two proteins, called bait and prey,
coupled to two halves of a transcription factor and expressed in
yeast.
• If the proteins make contact, they reconstitute a transcription
factor that activates a reporter gene.
Animation
10. • Used to detect interactions between candidate proteins whose
genes are available by constructing the appropriate hybrids and
testing for reporter gene activity
• Point mutations can be assayed to identify specific amino acid
residues critical for the interaction
• Can be used to screen libraries of activation domain hybrids to
identify proteins that bind to a protein of interest
• Transcriptional activation domains are commonly derived from the
Gal4 protein or the herpes simplex virus VP16 protein
• Reporter genes include the E. coli lacZ gene and selectable yeast
genes such as HIS3 and LEU2
11. Uses/ Advantages of Yeast Two Hybrid Method
• It is highly sensitive, detecting interactions that are not detected by
other methods
• Minimal binding constant required to detect an interaction in two
hybrid system is on the order of 1 µM
• Interactions are detected within the native environment of the cell
and hence that no biochemical purification is required
• Study of oncogenes and tumor suppressors and the related area of
cell cycle control
Limitations
• limited to proteins that can be localized to the nucleus, which may
prevent its use with certain extracellular proteins
• Proteins must be able to fold and exist stably in yeast cells and to
retain activity as fusion proteins
• Interactions dependent on a posttranslational modification that does
not occur in yeast cells will not be detected
• Many proteins, including those not normally involved in transcription,
will activate transcription when fused to a DNA-binding domain
12. Yeast three hybrid• The three-hybrid system enables the detection of RNA-protein
interactions in yeast using simple phenotypic assays.
• It was developed in collaboration with Stan Fields laboratory (University
of Washington).
• The LexA DNA binding domain is fused to the MS2 coat protein to form
Hybrid Protein 1.
• Hybrid Protein 2 consists of the Gal4 activation domain linked to the RNA
binding domain, Y, you wish to test.
• The Hybrid RNA consists of two MS2 RNA binding sites and the RNA
sequence you wish to test, RNAX.
• Hybrid Protein 1 and the presence of MS2 sites in the Hybrid RNA are
fixed, as is the Gal4 Activation Domain. RNAX and RNA Binding Domain Y
vary.
13. • This method consists in the expression in yeast cells of three chimerical
molecules, which assemble in order to activate two reporter genes.
• Thus, using the yeast three-hybrid system, in contrast to other methods,
RNA–protein interactions are detected in vivo.
• This system uses a transactivator protein in yeast, such as Gal4p, that is
able to recruit the transcriptional machinery and trigger transcription of a
gene.
• It consists of a DNA binding domain (DB) and an activation domain (AD)
and, importantly, these two domains are functionally independent,
meaning that they can be inserted into other molecules.
• In this method, the three components of the system are expressed from
two plasmids allowing the use of any previously described yeast strains
for two-hybrid system that provide the two reporter genes HIS3 and lacZ
under the control of a Gal4 operator.
14. Immunoprecipitation
• This is classical method of detecting protein-protein interactions
• Cell lysates are generated, antibody is added, the antigen is
precipitated and washed, and bound proteins are eluted and analyzed
• Coprecipitated protein is precipitated by the antibody itself and not by
a contaminating antibody in the preparation
• If the interaction is direct through another protein that contacts both
the antigen and the coprecipitated protein determining that the
interaction takes place in the cell.
• Adenovirus E1A protein interacts with Rb protein detects the
interactions present in a crude lysate
• Both the antigen and the interacting proteins are present in the same
relative concentrations as found in the cell.
• Co-immunoprecipitating proteins do not necessarily interact directly,
since they can be part of larger complexes
• Co-precipitation is not as sensitive as other methods, such as protein
affinity chromatography, because the concentration of the antigen is
lower than it is in protein affinity chromatography.
15. CO-IMMUNOPRECIPITATION (coIP)
• Co-immunoprecipitation (coIP) is the most complex method.
• Co-immunoprecipitation (co-IP) is a popular technique for protein
interaction discovery. Co-IP is conducted in essentially the same manner
as an immunoprecipitation (IP) of a single protein, except that the target
protein precipitated by the antibody, also called the "bait", is used to co-
precipitate a binding partner/protein complex, or "prey", from a lysate.
Animation
16. DATABASES
• Protein–protein interactions are only the raw material
for networks. To build a network, researchers typically
combine interaction data sets with other sources of
data. Primary databases that contain protein–protein
interactions include DIP (http://dip.doe-mbi.ucla.edu),
BioGRID, IntAct (http://www.ebi.ac.uk/intact) and
MINT (http://mint.bio.uniroma2.it).
• These databases have committed to making records
available through a common language called
PSICQUIC, to maximize access.
17. • A free, open-source database for archiving and exchanging
molecular assembly information. The database contains
Interactions, Molecular complexes and Pathways
• BIND Interaction Viewer Java showing how molecules can be
connected in the database from molecular complex to small
molecule. Yellow: protein, Purple: small molecule; white: molecular
complex;
• This session was seeded by the interaction between human LAT and
Grb2 proteins involved in cell signaling in the T-cell.
BIND: (Biomolecular Interaction Network Database)
18. • BIND Interaction Viewer Java
showing how molecules can be
connected in the database from
molecular complex to small
molecule.
• Yellow: protein
• Purple: small molecule;
• white: molecular complex;
• This session was seeded by the
interaction between human LAT
and Grb2 proteins involved in
cell signaling in the T-cell.
19. Study: Protein function, Protein-protein relationship, Evolution of
protein-protein interaction, The network of interacting proteins,
The environments of protein-protein interactions,
Predict: Unknown protein-protein interaction, The best interaction
conditions
• Identification numbers from :
SWISS-Prot, GenBank, PIR
DIP:(Database of Interacting Proteins)
20. The current status of DIP
• Number of proteins: 6978
• Number of organisms: 101
• Number of interactions:18260
• Number of distinct experiments describing an interaction: 22229
• Number of articles: 2203
Data Stored Data Format
BIND interactions
Molecular Complex
Pathways
ASN.1
XML
DIP interactions
Protein information
XIN
tab-delimited
21. Pathway Databases and Algorithms
• 1) KEGG (Kyoto Encyclopedia of Genes and Genomes): Representation
of higher order functions in terms of the network of interaction
molecules
• GENES database contains 240 943 entries from the published
genomes, including the bacteria, mouse and human.
• KEGG has 3 databases, GENES, PATHWAY and LIGAND databases.
• By matching genes in the genome and gene products in the pathway,
KEGG can be used to predict protein interaction networks and
associated cellular function.
• KEGG is a network of gene products with three types of interactions or
relations: enzyme-enzyme relations which catalyzes the successive
reaction steps in the metabolic pathway, direct protein-protein
interactions and gene expression relations.
• PATHWAY database contains 5761 entries including 201 pathway
diagrams with 14,960 enzyme-enzyme relations.
24. Protein Affinity Chromatography
• Affinity Chromatography is essentially a sample purification
technique, used primarily for biological molecules such as proteins.
• It is a method of separating a mixture of proteins or nucleic acids
(molecules) by specific interactions of those molecules with a
component known as a ligand, which is immobilized on a support.
• If a mixture of proteins is passed over (through) the column, one of
the proteins binds to the ligand on the basis of specificity and high
affinity (they fit together like a lock and key).
• The other proteins in the solution wash through the column because
they were not able to bind to the ligand.
• Protein fusions
– Glutathione S-transferase
– Staphylococcus protein A
– Maltose-binding protein
25. • The interactions of many
proteins with their target
proteins often depends on
the modification state of
one or both of the proteins
26. • Incredibly sensitive
– Can detect interactions with a binding constant as weak as
10-5
M
• Tests all proteins in an extract equally
• Easy to examine both the domains of a protein and the
critical residues within it that are responsible for a
specific interaction, by preparing mutant derivatives
• Interactions that depend on a multi subunit tethered
protein can be detected
• Independent criteria must be used to establish that the
interaction is physiologically relevant
27. Affinity Blotting
• Analogous to the use of affinity columns, proteins can be
fractionated by PAGE transferred to a nitrocellulose membrane,
and identified by their ability to bind a protein, peptide, or other
ligand
• Complex mixtures of proteins, such as total-cell lysates, can be
analyzed without any purification
• Biological activity of the proteins on the membrane, the
preparation of the protein probe, and the method of detection
widely used in studies of the regulatory subunit of the type II cAMP-
dependent protein kinase with numerous specific anchoring
proteins
• Two-dimensional procedures of isoelectric focusing followed by
SDS-PAGE have been used to increase the separation of proteins
28. Protein Probing
• A labeled protein can be used as a probe to screen an expression
library in order to identify genes encoding proteins that interact
with this probe.
• Interactions occur on nitrocellulose filters between an immobilized
protein and the labeled probe protein.
• The method is highly general and therefore widely applicable, in
that proteins as diverse as transcription factors and growth factor
receptors have been used as probe.
• The method is based on the approach of Young and Davis, who
showed that an antibody can be used to screen expression libraries
to identify a gene encoding a protein antigen
29. • The λgt11 libraries typically use an isopropyl-
b-D-thiogalactopyranoside (IPTG)-inducible
promoter to express proteins fused to b-
galactosidase.
• Proteins from the bacteriophage plaques are
transferred to nitrocellulose filters,
incubated with antibody, and washed to
remove non specifically bound antibody.
Protein probe can be manipulated in vitro to
provide, a specific posttranslational
modification or a metal cofactor
• Any protein or protein domain can be
specifically labeled for use as a probe
30. • Chromatin immunoprecipitation is a common technique for studying
epigenetics, as it allows the researcher to capture a snapshot of
specific protein–DNA interactions.
• ChIP include crosslinking the DNA and the protein in live cells, then
extracting and shearing the chromatin.
• Finally, the samples are immunoprecipitated with an antibody
targeting the protein of interest. The DNA is extracted from the
protein and can be evaluated either at specific regions of the genome
by quantitative PCR (qPCR)
Choosing an antibody for ChIP
• Monoclonal, oligoclonal (pools of monoclonals) and polyclonal
antibodies all can work in ChIP.
• monoclonal antibody is that generally it is more specific but
oligoclonal and polyclonal antibodies are better candidates for
recognizing target proteins, as they recognize multiple epitopes of the
targets.
Chromatin immunoprecipitation (ChIP)
31. • There are mainly two types of ChIP, primarily differing in the starting
chromatin preparation. The first uses reversibly cross linked chromatin
sheared by sonication called cross-linked ChIP (XChIP). Native ChIP (NChIP)
uses native chromatin sheared by micrococcal nuclease digestion.
Cross-linked ChIP (XChIP)
• Cross-linked ChIP is mainly suited for mapping the DNA target of transcription
factors or other chromatin-associated proteins, and uses reversibly cross
linked chromatin as starting material.
• The agent for reversible cross-linking could be formaldehyde or UV light.
• The DNA associated with the complex is then purified and identified by PCR,
microarray, molecular cloning and sequencing, or direct high-throughput
sequencing (ChIP-seq).
Native ChIP (NChIP)
• Native ChIP is mainly suited for mapping the DNA target of Histone modifiers.
Generally, native chromatin is used as starting chromatin.
• Then the chromatin is sheared by micrococcal nuclease digestion, which cuts
DNA at the length of the linker, leaving nucleosomes intact and providing DNA
fragments of one nucleosome (200bp) to five nucleosomes (1000bp) in length.
32. • Controls are essential for ChIP so, we need a “no-antibody control” (mock
IP) for each IP we are doing.
• To Know a region of DNA that should be enriched in IP and be amplified by
qPCR or Not??
Step 1: Crosslinking
• ChIP assays begin with covalent stabilization of the protein–DNA
complexes.
• As there is constant movement of proteins and DNA, ChIP captures a
snapshot of the protein–DNA complexes that exist at a specific time.
• In vivo crosslinking covalently stabilizes protein–DNA complexes and
performed using a formaldehyde solution.
Step 2: Cell lysis: In this step, cell membranes are dissolved with detergent
based lysis solutions to liberate cellular components, and crosslinked
protein–DNA complexes are solubilized.
• Because protein–DNA interactions occur primarily in the nuclear
compartment, removing cytosolic proteins can help reduce background
signal and increase sensitivity. Protease and phosphatase inhibitors are
essential at this stage to maintain intact protein–DNA complexes.
33. Step 3: Chromatin preparation (shearing/digestion): The extraction step yields
all nuclear material, which includes unbound nuclear proteins, full-length
chromatin, and the crosslinked protein–DNA complexes.
• DNA fragmentation is by sonication or enzymatically by digestion with
micrococcal nuclease.
• Ideal chromatin fragment sizes range from 200 to >700 bp; however, DNA
shearing is one of the most difficult steps to control. Sonication provides
truly randomized fragments, but limitation is Difficulty in maintaining
temperature during sonication.
Step 4: Immunoprecipitation
• To isolate a specifically modified histone, transcription factor, or cofactor of
interest, ChIP-validated antibodies are used to immunoprecipitate and isolate
the target from other nuclear components. This step eliminates all other
unrelated cellular material.
• Selection of the appropriate antibody is critical for successful ChIP assays.
Numerous ChIP-validated antibodies are available. For target proteins for which
qualified antibodies are unavailable, proteins fused to affinity tags such as HA,
Myc, His, T7, V5, or GST can be expressed in the biological samples, and then
antibodies against the affinity tags can be used to immunoprecipitate the
targets.
34. Step 5: Reversal of crosslinking, and DNA clean-up
• The Reversal of crosslinking and DNA clean-up done
through extensive heat incubations or digestion of the
protein component with Proteinase K.
• Treatment with RNase A is recommended as well to
obtain a more pure DNA sample.
• A final purification of the DNA from any remaining
proteins should be performed using phenolchloroform
extraction or spin columns designed for DNA purification.
Step 6: DNA quantitation
• We can quantitate the purified DNA products by qPCR.
• qPCR enables analysis of target protein–DNA complex
levels in different experimental conditions.
• There is a direct correlation between the amounts of
immunoprecipitated complex and bound DNA.