Western blot analysis of extracts from Jurkat cells, untreated or etoposide-treated (25 µM), using Cleaved DFF45 (Asp224) Antibody.
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 BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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 October 2016
Protocol Id: 10
Cleaved DFF45 (Asp224) Antibody detects the carboxy-terminal fragment of DFF45 resulting from cleavage at aspartic acid 224. The antibody does not recognize full length DFF45 or other cleavage products of DFF45.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to carboxy-terminal residues surrounding Asp224 of human DFF45. Antibodies are purified by protein A and peptide affinity chromatography.
Human DFF45 and its mouse homologue ICAD function in normal cells as chaperones for caspase-activated deoxyribonuclease (DFF40 or CAD) during its synthesis (1). The association of DFF45 (or its isoform DFF35) with DFF40 inhibits the DNAse activity of the latter (1-4). In vitro, DFF45 has been shown to be the target of several caspases, including caspase-3, -6, -7, -8 and granzyme B (3). In vivo, caspase-3 is believed to be the primary enzyme responsible for processing DFF45 and release of its carboxy-terminal fragment (3,5). The cleavage of DFF45 inactivates its inhibitory function on DFF40 and causes nuclear DNA degradation by DFF40, leading to cell death (6,7).
(This product is sold under license from Promega Corp., U.S. Patent No. 6,350,452.)
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