Western Blot analysis of HT29 cell extracts untreated, nocodazole-treated and lambda phosphotase-treated using Phospho-cdc25C (Thr48) (upper), and cdc25C (5H9) Rabbit mAb, #4688 (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-cdc25C (Thr48) Antibody detects endogenous levels of cdc25C only when phosphorylated at Thr48.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr48 of human cdc25C. Antibodies are purified by protein A and peptide affinity chromatography.
Cdc25 is a protein phosphatase responsible for dephosphorylating and activating cdc2, a crucial step in regulating the entry of all eukaryotic cells into mitosis (1). cdc25C is constitutively phosphorylated at Ser216 throughout interphase by c-TAK1, while phosphorylation at this site is DNA damage-dependent at the G2/M checkpoint (2). When phosphorylated at Ser216, cdc25C binds to members of the 14-3-3 family of proteins, sequestering cdc25C in the cytoplasm and thereby preventing premature mitosis (3). The checkpoint kinases Chk1 and Chk2 phosphorylate cdc25C at Ser216 in response to DNA damage (4,5).
Full activation of cdc25C involves phosphorylation at more than 12 different sites by cdc2/cyclin B and Polo-like kinase, and the activity of Pin1, a peptidyl-prolyl isomerase (PPI) (6,7). Pin1 contains a WW domain that binds phospho-Ser/Thr-Pro sites and a catalytic PPI region that induces a cis/trans isomerization on phospho-Ser/Thr-Pro bonds (8). Thr48 and Thr67 of cdc25C interact directly with the WW domain of Pin1 when these sites are phosphorylated (9). Thr48 phosphorylation also mediates binding to CKS/p13SUC1 (10).
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