Western blot analysis of untreated and UV-treated (50 mJ/cm2,30 min) HeLa cells and HT-1376 cells, using Phospho-BRCA1 (Ser1524) Antibody (upper) and BRCA1 Antibody #9010 (lower).Learn more about how we get our images.
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
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.
Phospho-BRCA1 (Ser1524) Antibody detects endogenous levels of BRCA1 only when phosphorylated at Ser1524.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser1524 of human BRCA1. Antibodies are purified by protein A and peptide affinity chromatography.
The breast cancer susceptibility proteins BRCA1 and BRCA2 are frequently mutated in cases of hereditary breast and ovarian cancers and have roles in multiple processes related to DNA damage, repair, cell cycle progression, transcription, ubiquitination, and apoptosis (1-4). BRCA2 has been shown to be required for localization of Rad51 to sites of double stranded breaks (DSBs) in DNA, and cells lacking BRCA1 and BRCA2 cannot repair DSBs through the Rad51-dependent process of homologous recombination (HR) (5). Numerous DNA damage-induced phosphorylation sites on BRCA1 have been identified, including Ser988, 1189, 1387, 1423, 1457, 1524, and 1542, and kinases activated in a cell cycle-dependent manner, including Aurora A and CDK2, can also phosphorylate BRCA1 at Ser308 and Ser1497, respectively (6-10). Cell cycle-dependent phosphorylation of BRCA2 at Ser3291 by CDKs has been proposed as a mechanism to switch off HR as cells progress beyond S-phase by blocking the carboxy terminal Rad51 binding site (11).
In Xenopus, in response to DNA damage, ATR-dependent and Claspin-mediated recruitment of BRCA1 leads to phosphorylation at Ser1524 (12).
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