Chromatin Immunoprecipitation (ChIP) Assays

Question 1

what is ChIP ?

Answer 1

Chromatin immunoprecipitation is an antibody-based technology used to selectively enrich specific DNA-binding proteins along with their DNA targets

Question 2

what is ChIP ?

Answer 2

Chromatin immunoprecipitation is an antibody-based technology used to selectively enrich specific DNA-binding proteins along with their DNA targets

Question 3

what are the 5 stages of ChIP ?

Answer 3

1. Crosslink
2. Chromtin Fregmentation
3. Immunoprcipitation
4. DNA Purification
5. DNA Analysis

Question 3

what are the 5 stages of ChIP ?

Answer 3

1. Crosslink
2. Chromtin Fregmentation
3. Immunoprcipitation
4. DNA Purification
5. DNA Analysis

Question 4

what are the 2 types of ChIP ?

Answer 4

• cross-linked ChIP (XChIP)
• Native ChIP (NChIP)

Question 5

how does XChIP use ?

Answer 5

uses reversibly cross-linked chromatin sheared by sonication

Question 6

what does NChIP use ?

Answer 6

uses netive chromatin shared by micrococcal nuclease digestion

Question 6

what does NChIP use ?

Answer 6

uses netive chromatin shared by micrococcal nuclease digestion

Question 7

what does Cross-linked ChIP (XChIP) do ?

Answer 7

• for mapping the DNA targetof transcription factors or other chromatin-associated proteins using reversibly cross-linked chromatin as starting material
• The agent for reversible cross-linking could be formaldehyde or UV light
• The cross-linked chromatin is usually sheared by sonication, providing fragments of 300 - 1000 base pairs (bp) in length
• Mild formaldehyde crosslinking followed by nuclease **digestion **has been used to shear the chromatin
• 400 - 500bp have proven to be suitable for ChIP assays as they cover two to three nucleosomes

Question 8

what do Cross-linked ChIP (XChIP) do ?

Answer 8

• Cell debrisin the sheared lysate is then cleared by sedimentation and protein–DNA complexes are selectively immunoprecipitated using specific antibodies to the protein(s) of interest
• The antibodies are commonly coupled to agarose, sepharose, or magnetic beads.
• The **immunoprecipitated complexes are then collected **and washed to remove non-specifically bound chromatin
• The protein–DNA cross-link is reversed and proteins are removed by digestion with proteinase K
• The DNA associated with the complex is then purified and identified by polymerase chain reaction (PCR), microarrays (ChIP-on-chip), molecular cloning and sequencing, or direct high-throughput sequencing (ChIP-Seq)

Question 9

1. DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP)
2. The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion
3. Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody
4. The associated DNA fragments are purified and their sequence is determined
5. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo

Answer 9

1. DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP)
2. The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion
3. Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody
4. The associated DNA fragments are purified and their sequence is determined
5. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo