Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 177

16. Wash the beads four times with Wash Buffer.
17. Resuspend the beads in 400 ml of Elution Buffer and incubate
in a water bath at 65-67°C for hours with occasional mixing.
18. Subject the sample to centrifugation (10,000 rpm, 30 s) to
pelletize the beads before removal.
19. Incubate the supernatant in a water bath at 65-67°C overnight
to enable the detachment of the protein and DNA fragments.
20. Centrifuge the sample again (10,000 rpm, 3 min) to remove
any residual beads.
21. The DNA in the supernatant can be isolated by adding 500 ml
of phenol/chloroform/isoamyl alcohol (25:24:1), vortex
thoroughly followed by centrifugation (14,000 rpm, 3 min)
enabling the portioning of two phases.
22. After carefully removing the aqueous phase, the organic phase
is mixed with 100 ml of 10 mM Tris, 1 mM EDTA, pH 8.1,
vortex thoroughly again and centrifuged at 14,000 rpm for
3 min to back extract DNA into the aqueous phase.
23. Pooled the aqueous phases and the DNA can be concentrated
by any commercial kits.
24. The DNA isolated is then sequenced using random hexamers
as primers.

Apart from identifying the DNA sequence, a specifi c protein or
groups of proteins can bind to, chIP can be extended to examine
modifi cation on DNA itself in regulating protein expression.
Recently, chips comprising arrays of defi ned DNA sequences are
used as a template for protein conjugated to fl uorophore to bind.
From the locale on the chip where the protein binds, the binding
DNA sequence is known. In view of the many parameters that
needed to be optimized, it is recommended that the pursuit of this
technique should start from commercially available kits that meet
the objectives of the study.

6.3. Variations

References

1. Burnette WN (1981) “Western blotting”: elec-
trophoretic transfer of proteins from sodium
dodecyl sulfate-polyacrylamide gels to unmod-
ifi ed nitrocellulose and radiographic detection
with antibody and radioiodinated protein A
(Translated from Eng). Anal Biochem
112(2):195-203 (in eng)
2. Garner MM, Revzin A (1981) A gel electro-
phoresis method for quantifying the binding of
proteins to specifi c DNA regions: application
to components of the Escherichia coli lactose
operon regulatory system (Translated from

Eng). Nucleic Acids Res 9(13):3047-3060
(in Eng)
3. Fried M, Crothers DM (1981) Equilibria and
kinetics of lac repressor-operator interactions
by polyacrylamide gel electrophoresis (Tran-
slated from Eng). Nucleic Acids Res 9(23):
6505-6525 (in Eng)
4. Gilmour DS, Lis JT (1984) Detecting protein-
DNA interactions in vivo: distribution of RNA
polymerase on specifi c bacterial genes
(Translated from Eng). Proc Natl Acad Sci U S A
81(14):4275-4279 (in Eng)