Western blot analysis of extracts from various cell types using Ape1 Antibody.
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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
Ape1 Antibody detects endogenous levels of total Ape1 protein.Species Reactivity:
Human, Mouse, RatSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino acids surrounding Ala230 of human Ape1. Antibodies are purified by peptide affinity chromatography.
Ape1 (Apurinic/Apyrimidic eEndonuclease 1), also known as Ref1 (Redox effector factor 1), is a multifunctional protein with several biological activities. These include roles in DNA repair and in the cellular response to oxidative stress. Ape1 initiates the repair of abasic sites and is essential for the base excision repair (BER) pathway (1). Repair activities of Ape1 are stimulated by interaction with XRCC1 (2), another essential protein in BER. Ape1 functions as a redox factor that maintains transcription factors in an active, reduced state but can also function in a redox-independent manner as a transcriptional cofactor to control different cellular fates such as apoptosis, proliferation and differentiation (3). Increased expression of Ape1 is associated with many types of cancers including cervical, ovarian, prostate, rhabdomyosarcomas and germ cell tumors (4). Ape1 has been shown to stimulate DNA binding of several transcription factors known to be involved in tumor progression such as Fos, Jun, NF-κB, PAX, HIF-1, HLF and p53 (4). Mutation of the Ape1 gene has also been associated with amyotrophic lateral sclerosis (ALS) (5,6).
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