Interaction Between Proteins and DNA

Chromatin Immunoprecipitation (ChIP)

Antigen vs Antibody

Antibody Structure & Isotype

Polyclonal Antibody: antibody preparations from immunized animals contain different antibodies produced by many B cell clonesMonoclonal Antibody:homogeneous antibody preparations produced in laboratoryconsist of a single type of antigen binding site (sing

How to make monoclonal antibodies?

Monoclonal Recombinant antibody

Nomenclature of Antibody

How to make phosphospecific antibodies?

Question

Protein A, G and L Antibody-Binding Ligands

ChIP-PCR/qPCR

ChIP on chip

ChIP-Seq

ATAC-Seq

ChIP-seq vs ATAC-seq

DNase Seq

MNase-seq

FAIRE-seq

Chromatin Structure Analyses

Transposase-Accessible Chromatin with Visualization (ATAC-see)

Luciferase Reporter Assay for mRNA Promoter

Electrophoretic Mobility Shift Assay (EMSA)

DNA Footprinting Assay

DNA Pull-Down Assay

Mass Spectrometry

Tandem Mass Spectrometry

enChIP

ENCODEEncyclopedia of DNA Elements

Applications: Protein-protein interaction

Protein Tag

GST (glutathione S-transferase) pull-down assay: a technique to test interaction between a tagged protein or the bait (GST, His6, biotin ...) and another protein (test protein, or prey)

Immunoprecipitation

Western Blotting

Consideration of Choosing Antibodies

Protein Position is different from what is expected

Immunohistochemistry (IHC)

Multiplexed protein detection (up to 96 proteins) on FFPE tissue

Used to discover protein-protein interactions by testing for physical interactions between two proteinsThe transcription factor is split into two separate fragments: The BD (binding domain): binding to the upstream activating sequence (UAS)Fused with bait