Question

1. Describe how a ChIP assay works:
•   What basic steps are involved in a ChIP assay?
•   What 3 (or 4) important pieces of information can this assay provide about a protein?
•   What are the limitations of this technique (eg. resolution)?
•   What sort of controls should be included and why?

Answer 1

ChIP means Chromatin immunoprecipitation. It is a powerful and versatile technique used for probing protein DNA interactions within the natural chromatin context of the cell(1,2). Cells are then lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion.It aims to determine whether specific proteins are associated with specific genomic regions,such as transcription factors on promoters or other DNA binding sites, and possibly defining cistromes. ChIP aims to determine the specific location in the genome that various histone modifications are associated with ,indicating the target of the histone modifiers.

* The basic steps are involved in a ChIP assay are:

step 1: Crosslinking

ChIP assays begin with covalent stabilization of protein -DNA complexes. Many protein -DNA interactions are transient and involve multi-protein complexes to orchestrate biological function.

step 2: Cell lysis

The lysis stage extracts the crosslinked protein-DNA complexes from cells or tissue and brings them into solution. At this stage, cellular components are liberated by dissolving the cell membrane with detergent-based solutions.

Step 3: Chromatin preparation

The extraction step yields all nuclear material, which includes unbound nuclear protein, full length chromatin and the crosslinked protein - DNA complexes.

Step 4: Immunoprecipitation

This step selectively enriches for the protein-DNA complex of interest and eliminates all other unrelated cellular material.

Step 5: Crosslinking reversal and DNA clean-up

Enrichment of DNA bound to the protein of interest is the goal forchromatin immunoprecipitation.

Step 6: DNA quantitation

Here have a direct correlation between the amounts of immunoprecipitated complex and bound DNA.

* Important pieces of information can this assay provide about a protein

1. DNA and associated proteins on chromatin in living cells or tissues are crosslinked.

2.The DNA protein complexes are then shared into ~500 bp DNA fragments by sonication.

3. Cross-linked DNA fragments associated with the protein of interest are selectively immunoprecipitated from the cell debris using an appropriate protein specific antibody.

* Limitations of this Technique

* Large Scale assays using ChIP is challenging using intact model organisms. This is because antibodies have to be generated for each TF , or, alternatively, transgenic model organisms expressing epitope-tagged TFs need to be produced.

* Researchers studying differential gene expression patterns in small organisms also face problems as genes expressed at low levels, in a small number of cells, in narrow time window.

* Chip experiments cannot discriminate between different TF isoforms.

ChIP -exo , a technique that adds exonuclease treatment to the ChIP process to obtain up to single base pair resolution of binding sites.

* Sort of controls included in ChIP assay

Immune function involves the tight control of transcription by the interplay of cis-regulatory elements and trans-acting transcription factors, which is largely influenced by the epigenetic landscape of immune cells.Previous studies have identified numerous transcription factors and chromatin modifiers implicated in innate and adaptive immunity. Recent technical advances in global techniques such as DNA microarrays and next generation sequencing allow analysis of entire genomes.