Immunoprecipitation Flashcards
What is immunoprecipitation?
Immunoprecipitation precipitates DNA using CENTRIFUGATION (ceramic beads to weigh it down as the pellet) or MAGNETISM (magnetic beads to anchor it) to isolate and concentrate the protein of interest (POI)
What are the types of immunoprecipitation? and what does it do?
Co-immunoprecipitation studies protein-protein interactions, Chromatin-immunoprecipitation studies genome sequences related to proteins, and RNA-immunoprecipitation studies the relationship between proteins and RNA. Co, chromatin, RNA.
Describe the process of chromatin-immunoprecipitation (ChIP).
FORMALDEHYDE crosslinks DNA and the amino groups of the protein of interest. Cell lysis by SONICATION OR ENZYME DEGRADATION releases the contents of the cell to get CHROMATIN FRAGMENTATION. IMMUNOPRECIPITATION is performed with a specific antibody then the immunoprecipitate is PURIFIED to remove the antibody via de-crosslinking. Then the ANALYSIS is performed by PCR/microarray/sequencing. Then the data is INTERPRETED with respect to the BIOLOGICAL ACTIVITY of the protein of interest.
Describe the process of ChIP-chip.
The protein of interest is selectively ChIP-ed then AMPLIFIED by PCR and fluorescently LABELLED. Then an aliquot of input DNA is labelled with another fluorophore. Both are MIXED and HYBRIDIZED onto a microarray. Analysis is performed to detect if the precipitated protein is bound to a target site which is seen when the intensity of the ChIP-DNA exceeds the input DNA.
What does the input lane tell you?
The input lane consists of housekeeping genes that all cells will have like scaffold proteins.
What is the difference between ChIP-sequencing and ChIP-chip?
ChIP-sequencing is directly processed into a DNA library to conduct bioinformatics analysis while ChIP-chip is processed onto a microarray first.
What are some applications of ChIP
? MCQ
Detect specific protein targets, binding sites and distribution of a protein, gene transcription & RNA polymerase activity, DNA and protein interactions, modifications to proteins, nucleosome architecture and regulation of chromosomal maintenance.
What is the process of RNA immunoprecipitation?
The cell is lysed to release RNA and their associated RNA-binding protein (RBP). Then there are 2 options to immunoprecipitate the RNA. Option 1 will prebind the antibody to magnetic beads and then bind the RBP to the antibody. Option 2 will directly bind the antibody to the RBP and then bind them to magnetic beads. The RNA immunoprecipitate is then washed to remove unbound antibodies and purified to de-crosslink the RNA from RBP and get the final RNA product that will be analysed on RT-qPCR or sequencing.
What are the 2 classes of RNA immunoprecipitation?
Native and cross-linked. Native is used to IDENTIFY RNA directly bound to protein and their ABUNDANCE in the sample. Crosslinked is used to precisely MAP the direct and indirect binding sites of the RBP.
What are the controls for ChIP?
Input DNA, no antibody control, isotype antibody control (IgM, IgG)
