Biology Reference
In-Depth Information
incubation period, less background, and less shearing forces on
the protein complexes in the protein lysate where centrifugation
impart during immunoprecipitation.
6. Chromatin
Immuno-
precipitation
Chromatin Immunoprecipitation (chIP) is developed as a tech-
nique that can determine fragments of the chromatin that binds to
a specifi c protein. The availability of antibodies that can target and
immunoprecipitate the specifi c protein of interest is mandatory to
employ this technique. This assay can be used to identify protein
that binds to specifi c nucleic acid sequences or to determine
modifi cation on chromatin in response to changes in protein
expression just to name a few. In the following, a generalized
procedure is adapted as a guide toward the usage of this assay.
1. 6% Paraformaldehyde
2. PBS
3. 1 M glycine
4. Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40,
and protease inhibitor cocktail)
5. High Salt Lysis Buffer (1% NP-40, 0.5% sodium deoxycholate,
0.1% SDS, and protease inhibitor cocktail in PBS)
6. Elution Buffer (1% SDS, 0.1 M NaHCO 3 )
7. Wash Buffer (100 mM Tris (pH 8.0), 500 mM LiCl, 1%
NP-40, and 1% deoxycholate)
8. Proteinase K
9. Protein A conjugated to sepharose beads
10. Phenol/chloroform/isoamyl alcohol (25:24:1)
6.1. Material and
Instruments
6.1.1. Materials
1. Hemocytometer or any mammalian cell count device
2. Sonication System
3. Vortex mixer
6.1.2. Instruments
6.2. Procedures
1. This procedure is adapted from the chIP assay protocol of the
chIP kit commercially available from Santa Cruz Biotechnology.
And the objective of this assay is to obtain the DNA sequences
that bound to a specifi c protein.
2. Approximately 2 × 10 7 cells are needed for chromosome immu-
noprecipitation. In the case of adherent cells, they must be
trypsinized to remove from the substratum and centrifuged
(1,200 rpm, 3 min) to form the cell pellet needed with the
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