week 4: immunoprecipitation Flashcards
section 5 (21 cards)
What is immunoprecipitation (IP)?
A method to isolate and concentrate a specific protein from a complex mixture using antibody-based capture.
What are protein A and protein G?
Bacterial proteins that bind to different antibody subtypes; attached to beads to capture antibody-protein complexes.
What does Co-IP study?
Protein-protein interactions — isolates complexes where proteins bind together.
General steps of Co-IP?
Sample preparation (non-ionic detergents like NP-40, Triton X-100)
Pre-clearing (removing non-specific proteins with beads alone)
Incubate with antibody
Precipitate complexes
Wash
Elute and analyze (SDS-PAGE, Western blot, Mass Spectrometry).
What is an isotype control?
A non-specific antibody control used to measure background binding.
How can IP combine with affinity chromatography?
Antibodies bind the protein of interest, captured on protein A/G resin, washed, and eluted for analysis.
How are proteins analyzed post-purification?
SDS-PAGE, Western blotting (immunoblotting), and mass spectrometry.
What is ChIP used for?
Identifies DNA regions bound to specific proteins (e.g. transcription factors, histone modifications).
General steps of ChIP?
Crosslink proteins to DNA
Cell lysis & chromatin fragmentation (sonication or MNase)
Immunoprecipitation with antibody
Wash, elute, reverse crosslink
DNA cleanup and analysis.
Controls used in ChIP?
Input DNA (reference sample)
No-antibody control
Isotype antibody control
Histone H3 antibody control.
What methods are used to analyze ChIP DNA?
PCR & qPCR (targeted analysis)
ChIP-chip (microarray hybridization)
ChIP-seq (DNA sequencing)
ChIP-PET, ChIP-DSL, ChIP-BA, MeDIP (specialized variations).
Limitation of PCR/qPCR in ChIP?
Primer bias — limited to known target regions.
Advantage of ChIP-seq?
Genome-wide, unbiased mapping of protein-DNA interactions.
What is the “% input” method?
(IP DNA quantity ÷ input DNA quantity) × 100
What is the “fold enrichment” method?
(IP DNA ÷ background noise control)
What is RIP used for? (RNA Immunoprecipitation)
To study protein-RNA interactions in vivo.
Two types of RIP?
Native RIP: identifies bound RNAs and their abundance.
Cross-linked RIP: maps precise binding sites on RNA.
Example of Co-IP experiment?
Studying protein interactions in mRNA decapping complexes (e.g. 4E-T and P-body formation in HeLa cells).
Applications of ChIP?
Identify transcription factor binding sites
Study gene regulation
Map chromatin architecture
Investigate disease mechanisms.
