Detection of PAR/pADPr by Western Blot. Western blot analysis of Wehi cellsuntreated (H) and treated (T) with 25 μM Etoposidefor 4 hours at 37 °C. Cells were lysed in Tris-GlycineSDS sample buffer at the concentration of 1 x 107cells/ml, and 10 μl of clarified lysate were loadedper well of 4-20% Tris-Glycine gel. Proteins weretransferred onto an Immobilon FL membrane andribosylated proteins were detected using Trevigen'spolyclonal anti-PAR antibody (cat# 4336-APC-050)followed by an IR680-conjugated secondary antibody(Licor). The membrane was scanned using anOdyssey Infrared Imaging System (Licor).
Preparation and Storage
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
-20°C (manual defrost freezer)
Procedure for Immunoblotting using Peroxidase Detection:
Blotting buffer: 12 mM Tris base, 96 mM Glycine, and 15% MeOH.
Blocking solution: 5% (w/v) nonfat dry milk in TBS-0.1% Tween.
Antibody solution: 5% (w/v) nonfat dry milk, in TBS-0.1% Tween.
Transfer the electrophoresed proteins to a PVDF membrane and incubate the membrane
for 1 hour at room temperature in blocking solution.
Incubate the membrane overnight at 4°C in antibody solution containing a 1:1000 dilution of
anti-PAR rabbit polyclonal antibody. Empirical determination of primary antibody
concentration will be required for optimal results.
Wash the membrane at room temperature for 5 minutes with 3 changes of TBS-0.1%
Tween. Changing the membrane containers often reduces background.
Incubate the membrane at room temperature for 1 hour in antibody solution containing antirabbit
conjugated to horseradish peroxidase.
Empirical determination of secondary antibody concentration will be required for optimal
Wash the membrane for 5 minutes with 4 changes of TBS-0.1% Tween.
Develop peroxidase reaction using chemiluminescence.
Affar, E.B., et al. 1998. Immunodot blot method
for the detection of poly(ADP-ribose) synthesized in
vitro and in vivo. Anal Biochem 259:280-3.
Shah, G.M., et al. 1995. Methods for biochemical
study of poly(ADP-ribose) metabolism in vitro and
in vivo. Anal Biochem 227:1-13.
Gagné, J-P., et al. 2008. Proteome-wide identification
of poly(ADP-ribose) binding proteins and
poly(ADP-ribose)-associated protein complexes.
Nucleic Acids Res 36:6959-76.
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