Immunoprecipitation Flashcards
1
Q
Simplest way of immunoprecipitation
A
- complex of proteins, add antibody, complementary proteins binds to antibody, protein A/G beads bind to protein-antibody complex, precipitated through centrifugation or magnetism
- OR conjugated bead-antibody complex binds to protein of interest, precipitated through centrifugation or magnetism
2
Q
What is Co-immunoprecipitation (Co-IP) used for?
A
- Analyze protein–protein interactions
3
Q
What is Chromatin-immunoprecipitation (ChIP) used for?
A
- Investigate regions of genome associated with a protein
- Identify specific proteins associated with a particular region of the genome
4
Q
What is RNA-immunoprecipitation (RIP) used for?
A
- Study the physical association between individual proteins and RNA molecules in vivo
5
Q
The steps involved in Co-immunoprecipitation (Co-IP)
A
- Sample preparation (non-ionic detergents e.g., NP-40, Triton X-100)
- Pre-clearing (just beads)
- Antibody incubation (target antibody or isotype control antibody)
- transfection of plasmids expressing protein of interest
- lysate and wash
- add antibody-immobilised agarose/sepharose resin
- precipitate immune complexes and wash precipitates
- Elution and analysis of precipitate (low pH or high salt solution)
6
Q
What types of analyses are used for following Co-IP?
A
- SDS-PAGE
- Western blotting
- Mass Spectrometry
7
Q
Steps in Chromatin-immunoprecipitation (ChIP)
A
- DNA-protein cross-linking and harvest cells
- Cell lysis
- Sonication or enzyme digestion, fragmentation of chromatin
- Immunoprecipitation with specific antibody, creates immune precipitate (ChIP material)
- Wash, elution and cross-link reversal
- DNA cleanup (purification) and analysis of DNA
8
Q
What types of analyses are used following ChIP?
A
- PCR
- qPCR
- microarray
- sequencing
9
Q
ChIP and antibodies (Ab)
A
- ChIP requires a highly epitope-specific Ab that recognises protein/residues of interest in their native chromatin states or possible cross-linked formation
- Theoretically anything associated with chromatin can be ChIPed, if an antibody can be raised.
Commercially available antibodies are recommended
10
Q
Cross-linking in ChIP
A
- formaldehyde will crosslink amino groups close by, not far away ones
- formaldehyde binds to amino group of one molecule, forms a Schiff base
- which then covalently binds to nucleotide molecule (cytosine, guanine, adenine etc) to from crosslink
11
Q
Cell lysis & Sonication of DNA in ChIP
A
- formaldehyde crosslinked cells undergo cell lysis, breaks into strands of DNA with covalently-bonded proteins
- sonication or MNase digestion breaks strand into smaller fragments so it can be seen on agarose gel (smaller molecular weights)
12
Q
Immunoprecipitation step in ChIP
A
- antibody-bead complex is added
- protein of interest is immunoprecipitated together with the crosslinked DNA
- decrosslinking and Proteinase K digestion of proteins
- proteins are eluted and purified
13
Q
Controls of ChIP
A
- Input DNA: A chromatin sample processed parallel to the other samples but lacks the IP step
- No Ab control: A chromatin sample processed parallel to the other samples but immunoprecipitated without specific antibody
- Isotype Ab control: A chromatin sample processed parallel to the other samples and immunoprecipitated with an isotype Ab control (IgG or IgM)
- Histone H3 antibody: A chromatin sample processed parallel to the other samples and immunoprecipitated with anti-H3 ab
14
Q
Analysis of ChIP DNA
A
- Identification of DNA regions associated with the protein/modification of interest
- PCR & real-time PCR
- Genome-wide mapping of DNA binding proteins including DNA microarray (ChIP-chip) & sequencing (ChIP-seq)
15
Q
% Input Method
A
- IP sample / input x 100
16
Q
Fold Enrichment Method (signal to noise fold change)
A
- IP sample / IgG (noise)
17
Q
ChIP-chip approach
A
- Protein of interest is selectively ChIPed.
- ChIP-enriched DNA amplified by PCR & fluorescently labelled.
- An aliquot of purified input DNA is labelled with another fluorophore.
- Two samples are mixed & hybridized onto a microarray.
- Binding of the precipitated protein to a target site is inferred when intensity of the ChIP DNA significantly exceeds that of the input DNA on the array.
18
Q
ChIP-DSL Approach
A
- conventional chromatin immunoprecipitation
- total DNA and ChIP DNA undergo reverse crosslinking & DNA biotinylation, added to DSL oligo pool
- oligo annealing to DNA
- solid phase selection & non-annealed oligo washes
- ligation with Taq ligase
- T3 and T7 PCR amplification
- hybridisation to 40mer oligo array
19
Q
ChIP-Seq Approach
A
- Instead of hybridizing the ChIP DNA to a microarray, each sample is processed directly into a DNA library for sequencing and subsequent bioinformatics analysis
20
Q
ChIP applications (what they discover)
A
- DNA sequences occupied by specific protein targets
- The binding sites and distribution of a particular protein, such as transcription factor, throughout the entire genome, under specified cellular conditions
- Gene transcription and RNA polymerase activity
- Complex DNA/protein interactions underlying disease phenotypes
- Modification to protein, such as histones, that many influence chromatin structure and gene expression
- Nucleosome architecture and regulation of chromosomal maintenance
21
Q
2 main classes of RNA immunoprecipitation (RIP)
A
- Native – used to identify RNAs directly bound by the protein and their abundance in the sample.
- Cross-linked – used to precisely map the direct and indirect binding site of the RBP of interest to the RNA molecule.
22
Q
Steps in RNA immunoprecipitation (RIP)
A
- Lysis cell
- Option 1: Prebind antibody - user specified antibody binds with Xtra magnetic Protein A beads, complex targets RBPs
- Option 2: Direct antibody binding - antibody directly binds to target RBP, antibody-target complexes to beads
- wash RIPs and purify RNA
- identify and profile RNA through RT-qPCR or sequencing
