Immunoprecipitation Flashcards
what is the principle of immunoprecipitation?
- specific antibodies are used to bind to the antigen of interest
- the antibodies are then bound to protein A/G beads
- the protein beads can then be precipitated using centrifugation or magnetism
- this allows the protein of interest to be isolated from a mixture of proteins
what are the different types of immunoprecipitations?
- co-immunoprecipitation (Co-IP)
- analyze protein-protein interactions
- chromatin-immunoprecipitation (ChIP)
- investigate regions of genome associated with a particular region of the genome
- RNA-immunoprecipitation (RIP)
- study the physical association between individual proteins and RNA molecules in vivo
what is co-immunoprecipitation?
this is a technique used to analyze protein-protein interactions, this is done by isolating one protein and investigating the proteins that can be found attached to it.
what are the general steps involved in co-immunoprecipitation?
Sample preparation
Pre-clearing
Antibody incubation
Precipitation of protein/protein complexes
Washing
Elution and analysis of precipitate
sample preparation what is it
mild detergents are used to lyse cells, the proteins are then placed on ice and protease inhibitors are used to prevent protein degradation
pre-clearing what does it include
off-target ab/isotype control, as there can be proteins with similar properties to the target protein or proteins that will bind to antibodies non-specifically (this increases the accuracy/specificity of the immunoprecipitation.
antibody incubation step explanation
the antibodies can be preloaded to beads or after the antigen-antibody interaction, the solution should be agitated to ensure complete binding
precipitation of protein/protein complexes explain step
centrifugation to cause pelleting or magnetism
washing - explain step
this is to remove impurities from the proteins
elution and analysis of precipitate - explain step
- elution buffer is used to break electrostatic interaction between the target and antibody so that the protein can then be analysed
- this can be:
- SDS-PAGE
- Western blotting
- mass spectrometry
- tandem mass spectrometry can also be used to analyse the AA sequence of the protein
elution and analysis of precipitate - explain step
- elution buffer is used to break electrostatic interaction between the target and antibody so that the protein can then be analysed
- this can be:
- SDS-PAGE
- Western blotting
- mass spectrometry
- tandem mass spectrometry can also be used to analyse the AA sequence of the protein
biological example of a co-immunoprecipitation experiment:
eIF4E-transporter protein, 4E-T:
eIF4E-transporter protein, 4E-T what is it
- needed to know if 4E-T also bound to decapping factors; CNOT1, PATL1, LSM14 + DDX6
- it was found that 4E-T interacts with 3 of the 4 mRNA decapping and decay machinery in a manner that is independent of its binding to elF4E
pay attention to the western blotting seen on the right side of the image, might be required to read one and understand if it is a positive result ETC.
what is chromatin immunoprecipitation?
- it is a technique used to:
- investigate regions of genome associated with a protein
- identify specific proteins associated with a particular region of the genome
what are the problems with chromatin-immunoprecipitation?
- it is hard to complete
- it is prone to errors
what are chromatin immunoprecipitation 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 are the general steps of chromatin immunoprecipitation?
- cross-link and harvest cells - fix proteins to DNA, using formaldehyde
- cell lysis + chromatin fragmentation - this is to break the chromatin into manageable sizes
- immunoprecipitation - this is when the chromatin is bound to an antibody
- wash, elution and cross-link reversal - this purifies the solution and then once pure unbinds the chromatin from the antibody
- DNA cleanup and analysis - this is with DNA purification methods, it can then be analysed with: PCR, qPCR, microarray or sequencing techniques
antibodies - overview
- they require highly epitope-specific Ab that recognises protein/residues of interest in their native chromatin states or possible cross-linked formation
- some targets are more difficult to ChIP due to associations with other proteins/structures in vivo which can mask the epitopes
- theoretically anything associated with chromatin can be ChiPed, but only if an antibody can be raised for it
- commercially available antibodies are recommended
ChIP - cross-linking stage:
Formaldehyde which works by cross linking amino groups between two amino acids
ChIP - cell lysis + sonication of DNA:
cell lysis releases the chromatin from the cell and then sonication is used to break down the chromatin fragments; fragments are broken down to about 500bp size
Purification steps:
Analysis of ChIP DNA:
identification of DNA regions associated with the protein/modification of interest
PCR + qPCR - to look for target sequence
genome wide mapping of DNA binding proteins -DNA microarray + sequencing
PCR + qPCR - to look for target sequence , what are the limitations?
- use of primers bias towards the sequences of interest
- makes the identification of unknown binding sites unlikely.
what are the controls for ChIP:
- negative controls:
- input DNA:
- a chromatin sample is processed parallel to the other samples but lacks the immunoprecipitation step
- No Ab control:
- a chromatin sample processed parallel to the other samples but immunoprecipitated without specific antibody
- Isotype Ab control:
- input DNA:
