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.
SMARCAD1 Antibody recognizes endogenous levels of total SMARCAD1 protein. This antibody also cross-reacts with a protein of unknown origin at 40 kDa.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human SMARCAD1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
SMARCAD1 is a SWI/SNF-like chromatin remodeling protein that plays a critical role in the maintenance of heterochromatin domains following DNA replication and proper chromosome segregation during mitosis (1-3). SMARCAD1 can be found in association with transcription repressor KAP1, histone deacetylases HDAC1/2, and lysine methyltransferase G9a/GLP, which are recruited to sites of DNA replication by PCNA. These proteins facilitate deacetylation of histones and methylation of histone H3 Lys9 to re-establish heterochromatin domains, such as centromeric regions (1). SMARCAD1 also plays a role in double stranded DNA break repair by facilitating DNA end resection and the subsequent repair by homologous recombination (4). Loss of SMARCAD1 results in increased sensitivity to DNA damaging agents, suggesting a role in the maintenance of genome stability.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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