Western blot analysis of extracts from SH-SY5Y, Saos-2 and Jurkat cells using BACH1/BRIP1 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
BACH1/BRIP1 Antibody detects endogenous levels of human BACH1 protein.Species Reactivity:
BACH1/BRIP1 Antibody is produced by immunizing rabbits with a synthetic peptide corresponding to amino acids near the carboxy terminus of human BACH1. Antibodies are purified by Protein A and peptide affinity chromatography.
BACH1, also known as BRIP1 and FANCJ, is a DNA helicase involved in repair of DNA cross-links and double strand breaks (1-3). Interaction between phosphorylated BACH1 and BRCA1 is required for DNA damage-induced checkpoint signaling (3,4). Originally identified as a breast cancer susceptibility gene (1), the BACH1 gene is mutated in Fanconi anemia (5), a recessive disorder characterized by multiple congenital abnormalities, progressive bone marrow failure, and high cancer risk/predisposition. Research investigators have concluded that BACH1 interactions with BRCA1 and the presence of BACH1 mutations in patients with early onset breast cancer indicate that BACH1 may act as a tumor suppressor (6).
Phosphorylation of BACH1 at Thr1133 is thought to be involved in regulation of the replication checkpoint and is required for the interaction of BACH1 with TopBP1 (7).
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