Confocal immunofluorescent analysis of MCF7 cells using p21 Waf1/Cip1 (12D1) Rabbit mAb (Alexa Fluor® 594 Conjugate) (red) and Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #3465 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 594 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated p21 Waf1/Cip1 (12D1) Rabbit mAb #2947.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 182
p21 Waf1/Cip1 (12D1) Rabbit mAb (Alexa Fluor® 594 Conjugate) recognizes endogenous levels of total p21 protein. The antibody does not cross-react with other CDK inhibitors.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human p21 protein.
The tumor suppressor protein p21 Waf1/Cip1 acts as an inhibitor of cell cycle progression. It functions in stoichiometric relationships forming heterotrimeric complexes with cyclins and cyclin-dependent kinases. In association with CDK2 complexes, it serves to inhibit kinase activity and block progression through G1/S (1). However, p21 may also enhance assembly and activity in complexes of CDK4 or CDK6 and cyclin D (2). The carboxy-terminal region of p21 is sufficient to bind and inhibit PCNA, a subunit of DNA polymerase, and may coordinate DNA replication with cell cycle progression (3). Upon UV damage or during cell cycle stages when cdc2/cyclin B or CDK2/cyclin A are active, p53 is phosphorylated and upregulates p21 transcription via a p53-responsive element (4). Protein levels of p21 are downregulated through ubiquitination and proteasomal degradation (5).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.