Revision 5

#2947Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, IHC-P, IF-IC, FC-FP

REACTIVITY:

H Mk

SENSITIVITY:

Endogenous

MW (kDa):

21

Source/Isotype:

Rabbit IgG

UniProt ID:

#P38936

Entrez-Gene Id:

1026

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:50
Immunohistochemistry (Paraffin) 1:50
Immunofluorescence (Immunocytochemistry) 1:400 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #19399.

Specificity / Sensitivity

p21 Waf1/Cip1 (12D1) Rabbit mAb detects endogenous levels of total p21 protein. The antibody does not cross-react with other CDK inhibitors.

Species Reactivity:

Human, Monkey

Species predicted to react based on 100% sequence homology

Dog

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy-terminus of human p21.

Background

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).

  1. Pestell, R.G. et al. (1999) Endocrine Rev. 20, 501-34.
  2. Cheng, J. et al. (1999) EMBO J. 18, 1571-83.
  3. Flores-Rozas, H. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8655-9.
  4. Wang, Y. and Prives, C. (1995) Nature 376, 88-91.
  5. Sheaff, R.J. et al. (2000) Cell 5, 403-10.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: 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.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 5
#2947

p21 Waf1/Cip1 (12D1) Rabbit mAb

Western Blotting Image 1: p21 Waf1/Cip1 (12D1) Rabbit mAb Expand Image
Western blot analysis from control HeLa cells (lane 1) or p21 Waf1/Cip1 knockout HeLa cells (lane 2) using p21 Waf1/Cip1 (12D1) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the p21 Waf1/Cip1 knockout HeLa cells confirms specificity of the antibody for p21 Waf1/Cip1.
Western Blotting Image 2: p21 Waf1/Cip1 (12D1) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell types using p21 Waf1/Cip1 (12D1) Rabbit mAb.
Western Blotting Image 3: p21 Waf1/Cip1 (12D1) Rabbit mAb Expand Image
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® p21 Waf1/Cip1 siRNA II (+), using p21 Waf1/Cip1 (12D1) Rabbit mAb #2947 and α-Tubulin (11H10) Rabbit mAb #2125. The p21 Waf1/Cip1 (12D1) Rabbit mAb confirms silencing of p21 Waf1/Cip1 expression and α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of p21 Waf1/Cip1 siRNA.
Western Blotting Image 1: p21 Waf1/Cip1 (12D1) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (1.0 mg/mL) from MCF-7 cells using p21 Waf1/Cip1 (12D1) Rabbit mAb #2947. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Immunoprecipitation Image 1: p21 Waf1/Cip1 (12D1) Rabbit mAb Expand Image
Immunoprecipitation of p21 from human umbillical vein endothelial cells (HUVECs) using p21 Waf1/Cip1 (12D1) Rabbit mAb. Western blot detection was performed using the same antibody.
Immunohistochemistry Image 1: p21 Waf1/Cip1 (12D1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using p21 Waf1/Cip1 (12D1) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Immunohistochemistry Image 2: p21 Waf1/Cip1 (12D1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded HeLa cells, transfected with SignalSilence® Control siRNA (Unconjugated) #6568 (left) or SignalSilence® p21 Waf1/Cip1 siRNA II #6558 (right), using p21 Waf1/Cip1 (12D1) Rabbit mAb.
Immunofluorescence Image 1: p21 Waf1/Cip1 (12D1) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of MCF7 cells using p21 Waf1/Cip1 (12D1) Rabbit mAb (red) and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow Cytometry Image 1: p21 Waf1/Cip1 (12D1) Rabbit mAb Expand Image
Flow cytometric analysis of Daudi cells using p21 Waf1/Cip1 (12D1) Rabbit mAb (right) and Propidium Iodide (PI)/RNase Staining Solution #4087, compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.