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.
Claspin Antibody detects endogenous levels of total claspin protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino acids near the carboxy terminus of human claspin. Antibodies are purified by peptide affinity chromatography.
Originally identified in Xenopus (1), and later in human cells (2), claspin is a mediator of Chk1 signal transduction at the replication checkpoint and in response to DNA damage. Expression of claspin is cell cycle-regulated, with protein levels peaking at the S/G2 phase (2). Expression is negatively regulated by both proteosome- and caspase-mediated degradation (3), and stabilized by activation of Chk1 (4). Claspin is a chromatin-bound protein, and has been shown to interact with the PNCA complex in the absence of DNA damage (5). Following checkpoint activation it remains chromatin-bound but is released from the PCNA complex and is phosphorylated in an ATR-dependent manner. Phosphorylated claspin interacts with several components of the DNA damage response including BRCA1 (6) and Chk1 (7), leading to ATR-dependent phosphorylation on each of these proteins. Phosphorylated Rad17 has also been shown to bind to and regulate the phosphorylation of claspin (8). It has been proposed that claspin behaves as a tumor suppressor in come cases since down-regulation promotes apoptosis following genotoxic stress (2). Conversely, claspin seems to behave as an oncogene in other instances since overexpression promotes cellular proliferation (6). Upregulated claspin has been suggested to be a sensitive marker of abnormally proliferating cells (9).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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