Western blot analysis of extracts from NIH/3T3 cells, untreated or staurosporine-treated (1 µM), and Jurkat cells, untreated or etoposide-treated (25 µM), using PARP Antibody.
Western blot analysis of HEK293 cell extracts, untreated (-) or PARP knock-out (+), using PARP Antibody #9542 (upper) or β-actin (13E5) Rabbit mAb #4970 (lower).
|REACTIVITY||H M R Mk|
|MW (kDa)||89, 116|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
PARP Antibody detects endogenous levels of full length PARP1 (116 kDa), as well as the large fragment (89 kDa) of PARP1 resulting from caspase cleavage. The antibody does not cross-react with related proteins or other PARP isoforms.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the caspase cleavage site in PARP. Antibodies are purified by protein A and peptide affinity chromatography.
PARP, a 116 kDa nuclear poly (ADP-ribose) polymerase, appears to be involved in DNA repair in response to environmental stress (1). This protein can be cleaved by many ICE-like caspases in vitro (2,3) and is one of the main cleavage targets of caspase-3 in vivo (4,5). In human PARP, the cleavage occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (2,4). PARP helps cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (6).
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