Immuno-Precipitation Flashcards
What Is Immunoprecipitation?
methods for isolation of proteins and other biomolecules from cell or tissue lysates for the purpose of subsequent detection by western blotting and other assay techniques.
Give the steps for basic immuno-precipitation
- Add antibody specific for protein ( this is usually created by adding the protein into a mouse and gathering antibodies from immune response)
- Add beads with protein A – a substance that binds antibodies strongly, ( these beads can be fixed onto a column or loose.
- This causes immunoprecipitation through the formation of the complex anti-protein and protein
- Wash off excess and then add salt to disrupt bonds between protein A beads and antibody eluting sample protein
- Run western blotting to separate elution by weight additionally an antibody can be added to this again so we know which is our target protein
Why do we complete Immuno-precipitation within ICE?
to avoid lysosomes destroying proteins.
Explain the steps of Co-immunoprecipitation (Co-IP) - Analyze protein–protein interactions.
- Cell or tissue that expresses protein is chosen and lysed using mild non-ionic detergents ( NP-40, Triton X-100) as to not effect protein structure.
- Solution is washed and agrose/Sepharose beads with specific antibody bound is added and antibody incubation occurs.
- Precipitation of protein/protein complexes are fourmed on beads and are washed.
- Final wash with high salt or low PH to elute complexes
- Complexes are ran through western blotting to be identified through mass if known protein, unknown can be identified through mass spec.
Explain the steps for Chromatin-immunoprecipitation (ChIP)
- DNA protein crosslinking through formaldehyde.
- Cell lysis through mild detergent
- Fragmentation of complexes through Sonication or enzyme digestion
- Immunoprecipitation with specific antibody
- Wash elution- and reverse cross links through leaving just DNA
- Analysis of DNA through Microarrays, qPRC or sequencing
Why are Chip certified antibodies more expensive/ harder to make?
- Requires a highly epitope-specific Ab that recognises protein/residues of interest in their native chromatin states or possible cross-linked formation, often more expensive for chip antibodies.
- Some protein targets are more difficult to ChIP due to associations with other proteins/structures in vivo masking epitopes.
Explain how Formaldehyde Chip crosslinking works?
Formaldehyde can readily permeate cell walls and membranes, resulting in efficient cross-linking, i.e. the formation of covalent bonds between proteins and DNA.
1. First, formaldehyde reacts with a relatively strong nucleophile, most commonly a lysine ε-amino group from a protein.
2. This reaction forms a methylol intermediate that can lose water to yield a Schiff base (animine).
3. This then leads to a cross linked Lysine-cytosine
4. These crosslinks are conserved during sonication and cell lysis
What are the two types of RNA Immunoprecipitation (RIP)
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.
How do we set controls for CHIP Immunoprecipitation ?
Always include:
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
How would you analyse Immunoprecipitation data using DNA microarray (ChIP-chip)?
- 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 using fluresence from the the flurchrome attached to dna.
Colours can be green hybridisation of gene 1 red hybridisation of gene 2 or a mix
How would you analyse Immunoprecipitation data using ChIP-DSL? (DNA selection and ligation strategy using specific oligos)
- DNA immunoprecipitation of CHIP DNA and Total DNA
- Reverse crosslinking and Biotinylating because biotin binds strongly to solid surfaces
- DSL oligo’s are then added which anneal to DNA
- Non annealing DNA is washed away.
- DNA ligates through the action of Taq ligase catalyzing the formation of a phosphodiester bond between the 5´-phosphate and the 3´-hydroxyl of two adjacent DNA strands.
- T3 and T7 PCR amplification and hybridisation to microarray.
- DNA will hybridise and produce a fluorescence signal allowing high-throughput analysis of target genes with much improved specificity and sensitivity.
Why do we do CHIP, what are the applications?
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
What is proteins A role in Immunoprecipertation?
Beads often express protein A on their surface as it binds strongly to Antibodies.
What is biotin role in Immuno-precipitation?
Biotinylating because biotin binds strongly to solid surfaces within CHIP DSL, solid phase.
