A brief introduction to two techniques used to study protein interactions: Yeast two hybrid (Y2H) system and Chromatin immunoprecipitation(ChIP)
I hope it helps and please comment if I've made any mistakes.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
High throughput next generation sequencing and robust transcriptome analysis help with gene expression profiling, gene annotation or discovery of non-coding RNA.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
High throughput next generation sequencing and robust transcriptome analysis help with gene expression profiling, gene annotation or discovery of non-coding RNA.
Introduction
Transcriptome analysis
Goal of functional genomics
Why we need functional genomics
Technique
1. At DNA level
2.At RNA level
3. At protein level
4. loss of function
5. functional genomic and bioinformatics
Application
Latest research and reviews
Websites of functional genomics
Conclusions
Reference
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.
With the DNA sequences of more than 90 genomes completed, as well as a draft sequence of the human genome, a major challenge in modern biology is to understand the expression, function, and regulation of the entire set of proteins encoded by an organism—the aims of the new field of proteomics. This information will be invaluable for understanding how complex biological processes occur at a molecular level, how they differ in various cell types, and how they are altered in disease states. The term proteomics describes the study and characterization of a complete set of proteins present in a cell, organ, or organism at a given time.
In general, proteomic approaches can be used (a) for proteome profiling, (b) for comparative expression analysis of two or more protein samples, (c) for the localization and identification of posttranslational modifications, and (d) for the study of protein-protein interactions. The human genome harbours 26000–31000 protein-encoding genes; whereas the total number of human protein products, including splice variants and essential posttranslational modifications (PTMs), has been estimated to be close to one million. It is evident that most of the functional information on the genes resides in the proteome, which is the sum of multiple dynamic processes that include protein phosphorylation, protein trafficking, localization, and protein-protein interactions. Moreover, the proteomes of mammalian cells, tissues, and body fluids are complex and display a wide dynamic range of proteins concentration one cell can contain between one and more than 100000 copies of a single protein.
A rapidly emerging set of key technologies is making it possible to identify large numbers of proteins in a mixture or complex, to map their interactions in a cellular context, and to analyze their biological activities. Mass spectrometry has evolved into a versatile tool for examining the simultaneous expression of more than 1000 proteins and the identification and mapping of posttranslational modifications. High-throughput methods performed in an array format have enabled large-scale projects for the characterization of protein localization, protein-protein interactions, and the biochemical analysis of protein function. Finally, the plethora of data generated in the last few years has led to approaches for the integration of diverse data sets that greatly enhance our understanding of both individual protein function and elaborate biological processes.
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
Transcriptome analysis
Goal of functional genomics
Why we need functional genomics
Technique
1. At DNA level
2.At RNA level
3. At protein level
4. loss of function
5. functional genomic and bioinformatics
Application
Latest research and reviews
Websites of functional genomics
Conclusions
Reference
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.
With the DNA sequences of more than 90 genomes completed, as well as a draft sequence of the human genome, a major challenge in modern biology is to understand the expression, function, and regulation of the entire set of proteins encoded by an organism—the aims of the new field of proteomics. This information will be invaluable for understanding how complex biological processes occur at a molecular level, how they differ in various cell types, and how they are altered in disease states. The term proteomics describes the study and characterization of a complete set of proteins present in a cell, organ, or organism at a given time.
In general, proteomic approaches can be used (a) for proteome profiling, (b) for comparative expression analysis of two or more protein samples, (c) for the localization and identification of posttranslational modifications, and (d) for the study of protein-protein interactions. The human genome harbours 26000–31000 protein-encoding genes; whereas the total number of human protein products, including splice variants and essential posttranslational modifications (PTMs), has been estimated to be close to one million. It is evident that most of the functional information on the genes resides in the proteome, which is the sum of multiple dynamic processes that include protein phosphorylation, protein trafficking, localization, and protein-protein interactions. Moreover, the proteomes of mammalian cells, tissues, and body fluids are complex and display a wide dynamic range of proteins concentration one cell can contain between one and more than 100000 copies of a single protein.
A rapidly emerging set of key technologies is making it possible to identify large numbers of proteins in a mixture or complex, to map their interactions in a cellular context, and to analyze their biological activities. Mass spectrometry has evolved into a versatile tool for examining the simultaneous expression of more than 1000 proteins and the identification and mapping of posttranslational modifications. High-throughput methods performed in an array format have enabled large-scale projects for the characterization of protein localization, protein-protein interactions, and the biochemical analysis of protein function. Finally, the plethora of data generated in the last few years has led to approaches for the integration of diverse data sets that greatly enhance our understanding of both individual protein function and elaborate biological processes.
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.
The three hybrid system of yeast has been described in this ppt. Yeast one Hybrid system, yeast two hybrid system and yeast 3 hybrid system is explained. This explain about the DNA-protein interaction and Protein-DNA-Protein interaction.
Protein protein interaction, functional proteomicsKAUSHAL SAHU
IntroductionTypes of Protein-protein interactionsEffects of Protein-Protein InteractionsProtein-Protein Interaction Identification Methods :- Experimental (In vivo) Yeast two hybrid system- Experimental (In vitro) Co-immunoprecipitation, ChIP, Affinity Blotting, Protein Probing - Computational (In silico) Database of interacting proteins, VisANT etc.
ConclusionReferences
Current trends in pseduogene detection and characterizationShreya Feliz
This presentation gives the insight of the current trends in detecting and characterizing Pseudogenes. Pseudogenes detection by bioinformatics may enhance the understanding of Pseudogenes and take research to the next step.
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Interplay between Metabolism and EpigeneticsAbhishek M
This ppt gives you an idea about the inter-connections between epigenetics and metabolism. How nutrition affects epigenetics and how epigenetic changes may cause metabolic disorders.
Here is an introduction to the renal mechanisms of clearance and pH balance with some slides dedicated to the differences between metabolic and respiratory acidosis and alkalosis.
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Here is some information about 5 important immunological techniques including Flowcytometry and RIA
I hope it helps and please comment if u come across any mistakes or scope for improvement, it'll really be appreciated.
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 .
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This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
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Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
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A brief information about the SCOP protein database used in bioinformatics.
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The yeast two hybrid system and ChIP
1. The Yeast Two Hybrid
System
The technique was pioneered by Stanley
Fields and Ok-kyu Song in 1989.
It is an important technique used to study
protein-protein interactions.
This procedure is based on the discovery that
gene expression in S. cerevisiae depends on
interactions between pairs of transcription
factors or proteins.
2. A protein is composed of domains, which
allow the same protein to perform different
functions.
The Y2H system uses two protein domains
that have specific functions.
A DNA binding domain(BD) capable of
binding to DNA
An activation domain(AD) capable of
activating transcription of DNA.
3. The assay relies on the expression of a
reporter gene (such as lacZ or GFP), which
is activated by the binding of a particular
transcription factor.
The query protein of interest fused with the
BD is known as the Bait, and the protein
library fused with the AD is referred to as
the Prey.
The successful interaction between the
proteins is linked to a change in the cell
phenotype.
4. In the absence of Bait-Prey interaction, the
AD domain is unable to localize to the
reporter gene to drive gene expression.
However, when Bait and Prey interact, the
BD domain binds to the DNA localizing the
AD domain upstream of the reporter gene,
leading to the expression of reporter gene.
6. RECOVERY OF
INFORMATION:
Once the selection has been performed, the
primary structure of the proteins which
display the appropriate characteristics must
be determined. This is achieved by retrieval
of the protein-encoding sequences (as
originally inserted) from the cells showing the
appropriate phenotype.
7. Advantages of Y2H
system:-
1. Its low tech, can be easily carried out in a
lab.
2. It helps in identification of interaction
partners.
3. Determination of crucial sequences for
interaction.
4.Determination of unknown protein function.
8. Limitations of Y2H system:-
1. A particular interaction may be absent in
yeast(like if a bacterial protein is tested, the
system may lack the chaperones important
for folding or interaction).
2. If test protein are not localized to the
nucleus, interacting proteins may show a
negative result.
3. Fusion proteins may inhibit the original
interaction.
9. Chromatin
Immunoprecipitation(ChI
P)
It is a type of immunoprecipitation
experimental technique used to
investigate the interaction between
proteins and DNA in the cell. It aims to
determine whether specific proteins are
associated with specific genomic regions,
such as transcription factors on promoters
or other DNA binding sites.
10. Procedure:
1) DNA and associated proteins on chromatin
in living cells or tissues are cross linked.
2) The DNA-protein complexes are then
sheared into ~500 bp DNA fragments by
sonication or nuclease digestion.
3) Cross-linked DNA fragments associated
with the protein(s) of interest are selectively
immunoprecipitated from the cell debris using
an appropriate protein-specific antibody.
11. 4)The associated DNA fragments
are purified and their sequence is
determined.
5)Enrichment of specific DNA
sequences represents regions on
the genome that the protein of
interest is associated with in vivo.
13. Types of ChIP :-
1. Cross-linked ChIP
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.
2. Native ChIP
Native ChIP is mainly suited for mapping the
DNA target of histone modifiers. Generally, native
chromatin is used as starting chromatin.
14. ChIP Limitations:
1)It cannot distinguish between transcription
factor isoforms.
2)Large scale assays are challenging, as
antibodies for each TF needs to be made.
3) ChIP does not distinguish between a single
protein and a complex.
4) The results from ChIP cannot be viewed or
analyzed directly, so further protocols such as
Polymerase Chain Reaction (PCR).