Western blot analysis of HT-29 cell extracts, untreated (-) or treated with thymidine (2 mM, 16 hr) followed by Nocodazole #2190 (10 nM, 24 hr; +), using Phospho-Cyclin B1 (Ser116) Antibody (upper), Cyclin B1 (D5C10) XP® Rabbit mAb #12231 (middle), or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).Learn more about how we get our images.
Western blot analysis of HT-29 cell extracts, untreated (AS) or synchronized in S-phase by double thymidine block (2 mM) followed by release into fresh medium for the indicated time, using Phospho-Cyclin B1 (Ser116) Antibody (upper), Cyclin B1 (D5C10) XP® Rabbit mAb #12231 (middle), or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).Learn more about how we get our images.
Western blot anaylsis of HeLa cell extracts, untreated (-) or treated with Nocodazole #2190 (10 nM, 24 hr; +) using Phospho-Cyclin B1 (Ser116) Antibody (upper), Cyclin B1 (D5C10) XP® Rabbit mAb #12231 (middle), or α-Actinin (D6F6) XP® rabbit mAb #6487 (lower). Membranes were mock treated (- CIP) or Calf Intestinal Alkaline Phosphatase treated (+ CIP) after transfer.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-Cyclin B1 (Ser116) recognizes endogenous levels of cyclin B1 protein only when phosphorylated at Ser116.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser116 of human cyclin B1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Cyclins are a family of proteins that activate specific cyclin-dependent kinases required for progression through the cell cycle. The entry of all eukaryotic cells into mitosis is regulated by activation of cdc2/cdk1 at the G2/M transition. This activation is a multi-step process that begins with the binding of the regulatory subunit, cyclin B1, to cdc2/cdk1 to form the mitosis-promoting factor (MPF). MPF remains in the inactive state until phosphorylation of cdc2/cdk1 at Thr161 by cdk activating kinase (CAK) (1,2) and dephosphorylation of cdc2/cdk1 at Thr14/Tyr15 by cdc25C (3-5). Five cyclin B1 phosphorylation sites (Ser116, 126, 128, 133, and 147) are located in the cytoplasmic retention signal (CRS) domain and are thought to regulate the translocation of cyclin B1 to the nucleus at the G2/M checkpoint, promoting nuclear accumulation and initiation of mitosis (6-9). While MPF itself can phosphorylate Ser126 and Ser128, polo-like kinase 1 (PLK1) phosphorylates cyclin B1 preferentially at Ser133 and possibly at Ser147 (6,10). At the end of mitosis, cyclin B1 is targeted for degradation by the anaphase-promoting complex (APC), allowing for cell cycle progression (11). Research studies have shown that cyclin B1 is overexpressed in breast, prostate, and non-small cell lung cancers (12-14).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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