Biology Reference
In-Depth Information
In turn, the Ad IVa2 protein is essential for virus assembly and the
formation of empty viral capsids [
6
]. Ad IVa2 is found in both empty
and mature virus particles. The IVa2 protein in vitro binds to packag-
ing A repeats 1 and 2 as well as A repeats 4 and 5 [
8
]. Furthermore,
our recent study suggests that another two Ad proteins, IIIa and
L4-22 K, can interact with the packaging sequences in vivo and are
required for effi cient virus encapsidation [
9
,
10
].
In this Chapter, we describe a chromatin immunoprecipitation
(ChIP) approach that was used to study the binding of the Ad IVa2,
L1-52/55 K as well as IIIa and L4-22 K proteins to wild type and
mutant packaging sequences in vivo using specifi c antisera directed
against these products [
11
]. The method represents adaptations
derived from protocols described previously [
12
-
14
]. Viral chro-
matin was sheared by sonication to an average size of ~200-500 bp
and the products of immunoprecipitation were quantifi ed using
real-time quantitative PCR (qPCR). This assay permits accurate
quantifi cation over a wide range of DNA concentrations. The use of
formaldehyde cross-linking to stabilize DNA-protein and protein-
protein complexes formed in vivo allows the identifi cation of
macromolecular complexes found in living cells.
2
Materials
2.1 Cells
and Medium
1. N52.E6 cells (which express Ad E1A and E1B proteins) [
15
].
2. Dulbecco's Modifi ed Eagle's Medium supplemented with
10 % bovine fetal serum and penicillin/streptomycin.
1. Formaldehyde: 37 % formaldehyde.
2. 1 M Glycine.
3. Phosphate-buffered saline (PBS).
4. Sodium dodecyl sulfate (SDS) lysis buffer: 50 mM Tris-HCl,
pH 8.0, 10 mM ethylene diamine tetraacetic acid (EDTA),
1 % SDS.
5. Protease inhibitors (working concentrations in SDS lysis buf-
fer,
item 6
): phenylmethylsulfonyl fl uoride (PMSF; 1 mM,
Sigma Cat. # P7626), aprotinin (1
2.2 Reagents
and Buffers
μ
g/
μ
l Sigma Cat. # A1153),
L Sigma Cat. # P4265).
6. IP dilution buffer: 16.7 mM Tris-HCl, pH 8.0, 167 mM
NaCl, 1.2 mM EDTA, 0.01 % SDS, 1.1 % Triton X-100.
7. High salt wash buffer: 20 mM Tris-HCl, pH 8.0, 500 mM
NaCl, 2 mM EDTA, 0.1 % SDS, 1 % Triton X-100.
8. Low salt wash buffer: 20 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 2 mM EDTA, 0.1 % SDS, 1 % Triton X-100.
9. LiCl wash buffer: 10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 1 mM
EDTA, 1 % SDS, 0.5 % Triton X-100, 1 % sodium deoxycholate.
and pepstatin A (1
μ
g/
μ
