Anti p21

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China

Anti-p21 is a primary antibody that specifically recognizes the p21 protein. p21 is a cyclin-dependent kinase inhibitor that plays a crucial role in cell cycle regulation. This antibody can be utilized for the detection and quantification of p21 in various experimental applications.

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Rabbit anti-p21 (28 citations) Mouse anti-p21 (10 citations) Anti-p21 (2947S, (9 citations) Rabbit anti-p21 Waf1/Cip1 (4 citations) Anti-HMGA2 and p21 (4 citations) Anti-CDKN1A/p21 (3 citations) Anti-p21 antibody (2947S) (3 citations) Rabbit anti-p21 antibody (2 citations) Anti-p21 and PARP antibodies (2 citations) Monoclonal rabbit anti-p21 waf1/cip1 (2 citations)

226 protocols using anti p21

Immunodetection of Protein Modifications

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The following primary antibodies were used: anti-FLAG (rabbit, F7425; Sigma-Aldrich; dilution used in WB: 1:3000), anti-FLAG (mouse, M2; Sigma-Aldrich; dilution used in ICC: 1:2000), anti-HA (rabbit, Y-11; Santa Cruz Biotechnology; dilution used in WB: 1:1000, ICC: 1:1000), anti-p21 (rabbit, 12D1; Cell Signaling Technology; dilution used in WB: 1:1000), anti-p21 (mouse, DCS60; Cell Signaling Technology; dilution used in immune-precipitation: 1:50), anti-PRMT6 (rabbit, D5A2N; Cell Signaling Technology; dilution used in WB: 1:1000), anti-α-Tubulin (mouse, DM1A; CALBIOCHEM; dilution used in WB: 1:1000), anti-histone H3 (rabbit, ab1791; abcam; dilution used in WB: 1:10000), anti-phosphor p21 (Thr 145) (rabbit, PA5–12646; Pierce; dilution used in WB: 1:1000). An anti-R156 dimethylated p21 antibody (Thermo Fisher Scientific; dilution used in WB: 1:500) was produced in rabbit immunized with a synthetic peptide.

Nakakido M., Deng Z., Suzuki T., Dohmae N., Nakamura Y, & Hamamoto R. (2015). PRMT6 increases cytoplasmic localization of p21CDKN1A in cancer cells through arginine methylation and makes more resistant to cytotoxic agents. Oncotarget, 6(31), 30957-30967.

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Antibody Characterization for Protein Analysis

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Mouse monoclonal anti-β-actin (catalogue no. FB075) antibody was obtained from Nanchang Focus Bioscience Co., Ltd, and the species specificity is human, mouse, rat and pig. Anti-E-cadherin (catalogue no. 14472) and Anti-p21 (catalogue no. 2947) antibodies were obtained from Cell Signalling Technology, Inc. (Danvers, MA, USA), the species specificity of Anti-E-cadherin is human, mouse and rat, and the species specificity of Anti-p21 is human and monkey. Anti-MORF4L1 (catalogue no. HPA042360) antibody was obtained from Sigma-Aldrich (Merck KGaA; Darmstadt, Germany), the species specificity of anti-MORF4L1 is human and rat.

Sang Y., Zhang R., Sun L., Chen K.K., Li S.W., Xiong L., Peng Y., Zeng L, & Huang G. (2018). MORF4L1 suppresses cell proliferation, migration and invasion by increasing p21 and E-cadherin expression in nasopharyngeal carcinoma. Oncology Letters, 17(1), 294-302.

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Immunoprecipitation and Western Blotting

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Collected cells were washed with cold PBS and lysed in ice-cold buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40] supplemented with complete protease inhibitors (Sigma-Aldrich) on ice for 10 min. The samples were centrifuged at 13,000rpm for 10 min. The lysates were aliquoted into two tubes and incubated with either the designated antibody or an appropriate IgG control for 16 hours at 4 °C. Protein A/G agarose (Invitrogen) was used to precipitate antibody-protein complexes.
For Western blotting, anti-CD24 (Santa Cruz Biotechnology, 1:1000), anti-p27 (Cell Signaling, 1:1000), anti-p21 (Cell Signaling, 1:1000), anti-p53 (Santa Cruz Biotechnology, 1:2000), anti-Lamin (Santa Cruz Biotechnology, 1:5000), anti-H1.5 (Abcam, 1:5000), anti-Cyclin B1 (Santa Cruz Biotechnology, 1:3000), anti-GFP (Cell Signaling, 1:2000), anti-NPM1 (Abcam, 1:3000), anti-HA (Sigma-Aldrich, 1:3000), anti-Myc (Abcam, 1:3000), anti-ARF (Santa Cruz Biotechnology, 1:1000), anti-MDM2 (Santa Cruz Biotechnology, 1:1000), and anti-β-actin (Sigma, 1:5000) antibodies were used as primary antibodies. The membranes were incubated for 1 hour at room temperature in 0.25% nonfat milk with a 1:3,000–5,000 dilution of either anti-rabbit or anti-mouse IgG HRP-linked secondary antibody (Cell Signaling).

Wang L., Liu R., Ye P., Wong C., Chen G.Y., Zhou P., Sakabe K., Zheng X., Wu W., Zhang P., Jiang T., Bassetti M.F., Jube S., Sun Y., Zhang Y., Zheng P, & Liu Y. (2015). Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation. Nature Communications, 6, 5909.

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Western Blot Analysis of Cell Signaling Proteins

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Protein sample preparation and western blot assay were performed as previously described (24 (link)). The primary antibodies used were: anti-MTMR3 (1:500; cat. no. 12443; Cell Signaling Technology, Inc.), anti-Cyclin D1 (1:1,000; cat. no. 60186-1-1g; ProteinTech Group, Inc.), anti-cyclin-dependent kinase 2 (1:1,000; CDK2; cat. no. 11026-2-AP; ProteinTech Group, Inc.), anti-p62 (1:1,000; cat. no. 19117-1-AP; ProteinTech Group, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-Cyclin E (1:1,000; cat. no. sc-247; Santa Cruz Biotechnology, Inc.), anti-Cyclin A (1:1,000; cat. no. sc-751; Santa Cruz Biotechnology, Inc.), anti-cell division cycle 25 A (1:1,000; cdc25A; sc-7157; Santa Cruz Biotechnology, Inc.), anti-microtubule associated protein 1 light chain 3 (LC3) A (1:500; cat. no. 4599; Cell Signaling Technology, Inc.), anti-LC3B (1:500; cat. no. 3868; Cell Signaling Technology, Inc.) and anti-GAPDH (1:20,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.). Bound HRP-labeled secondary antibody (1:5,000; cat. no. SC-2005 or SC-2054; Santa Cruz Biotechnology, Inc.) was assayed by super ECL detection reagent (Pierce; Thermo Fisher Scientific, Inc.). Protein density of western blots was analyzed using ImageJ 1.51k software (National Institutes of Health).

Wang Z., Zhang M., Shan R., Wang Y.J., Chen J., Huang J., Sun L.Q, & Zhou W.B. (2019). MTMR3 is upregulated in patients with breast cancer and regulates proliferation, cell cycle progression and autophagy in breast cancer cells. Oncology Reports, 42(5), 1915-1923.

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SDS-PAGE and Protein Immunoblotting

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Cells protein lysates were separated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22 μm NC membranes (Sigma), and incubated with specific antibodies. GAPDH antibody was used as control.
Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad). Anti-p15 was purchased from Santa Cruz Biotechnology, Inc, and anti-p21 was purchased from Cell Signaling Technology.

Chen W.M., Huang M.D., Sun D.P., Kong R., Xu T.P., Xia R., Zhang E.B, & Shu Y.Q. (2016). Long intergenic non-coding RNA 00152 promotes tumor cell cycle progression by binding to EZH2 and repressing p15 and p21 in gastric cancer. Oncotarget, 7(9), 9773-9787.

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Western Blot Analysis of Cellular Proteins

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Protein lysates were separated by 10% SDS-PAGE and then transferred to nitrocellulose membranes by electroblotting. The membranes were incubated with anti-IκB kinase, anti-GADPH, anti-BCL2, anti-BAX, anti-p21, or anti-β-ACTIN antibodies (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C before subsequent incubation with secondary antibodies conjugated with horseradish peroxidase for 1 h at 37 °C. Proteins were visualized using enhanced chemiluminescence reagent.

Huang C.P., Chen J., Chen C.C., Liu G., Zhang Y., Messing E., Yeh S, & Chang C. (2019). ASC-J9® increases the bladder cancer chemotherapy efficacy via altering the androgen receptor (AR) and NF-κB survival signals. Journal of Experimental & Clinical Cancer Research : CR, 38, 275.

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Western Blot Analysis of Cellular Proteins

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Total protein from cultured cells was extracted with RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein were subjected to Western blotting as described previously.4 (link),22 (link),23 (link) The primary antibodies used were as follows: anti-THAP7 was purchased from Sigma-Aldrich, anti-p21 was obtained from Cell Signalling Technology (Cambridge, MA, USA), and anti-GAPDH was purchased from Bioworld Company (Nanjing, China).

Chen C.P., Sang Y., Liu L., Feng Z.Q., Liang Z, & Pei X. (2019). THAP7 promotes cell proliferation by regulating the G1/S phase transition via epigenetically silencing p21 in lung adenocarcinoma. OncoTargets and therapy, 12, 5651-5660.

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Western Blot Analysis of Protein Expression

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Anti-FLAG and anti-beta actin (Sigma-Aldrich, USA); anti-GNL1, anti-RPS20, anti-CDK1 and anti-MDM2 (Abcam, UK); anti-GFP, anti-Cyclin B1 and anti-p53 (Santa Cruz, USA); anti-p21, anti-Cyclin D1, anti-CDK4, anti-pRbSer-780, anti-Rb and anti-E2F1 (Cell Signaling Technology Inc., USA) antibodies were used in western blot analysis for checking protein expression.

Krishnan R., Boddapati N, & Mahalingam S. (2018). Interplay between human nucleolar GNL1 and RPS20 is critical to modulate cell proliferation. Scientific Reports, 8, 11421.

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Overexpression and Silencing of PRSS8

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The full length of PRSS8 was cloned from human cDNA and inserted into pEGFP vector (Promega, Madison, WI), to generate pEGFP -PRSS8, expression constructs. A 19-nt siRNA oligonucleotide with 3′-dt extensions against human PRSS8 transcript and one scrambled siRNA (negative control) were designed, as shown in Supplementary Table S1. siRNAs were synthesized by Shanghai GenePharma Inc. (Shanghai, China). Twenty-four hours before transfection, 1.0×10⁵ cells were seeded in a 6-well plate. 4 μg of PRSS8 expression plasmid or negative control plasmid was transfected into cells, using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) following the manufacture's protocol. The cells were collected for immunoblotting analysis using the following primary antibodies: anti-PRSS8, anti-P21, anti-Cyclin D1, anti-E-cadherin, anti-snail and anti-Twist (from Cell Signaling Technologies Inc., CA). Anti-β-actin (Sigma, St Louise, MO) was used as internal loading control.

Bao Y., Wang Q., Guo Y., Chen Z., Li K., Yang Y., Zhang H., Dong H., Shen K, & Yang W. (2016). PRSS8 methylation and its significance in esophageal squamous cell carcinoma. Oncotarget, 7(19), 28540-28555.

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Comprehensive Antibody Panel for Cell Signaling Analysis

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Catalog numbers and dilution fold are listed in parentheses. The following antibodies were purchased from Cell Signaling Technology: Anti-Bak (3792; 1:1000), anti-Bax (2772; 1:1000), anti-Bcl-2 (2870; 1:1000), anti-Bcl-xL (2764; 1:500), anti-CHK1 (2360; 1:1000), anti-phospho-CHK1 (S345; 2348; 1:1000), anti-cleaved caspase-3 (9661; 1:1000), anti-cleaved caspase-7 (9491; 1:1000), anti-cleaved caspase-8 (9496; 1:500), anti-cleaved caspase-9 (9501; 1:1000), anti-cytochrome c (4272; 1:500), anti-H2A.X (7631; 1:1000), anti-phospho-H2A.X (S139; 9718; 1:1000), anti-p53 (2524; 1:1000), anti-p53 (2527; 1:1000), anti-phospho-p53 (S6; 9285; 1:1000), anti-phospho-p53 (S15; 9284; 1:1000), anti-p21 (2946; 1:1000), and anti-voltage-dependent anion channel (VDAC; 4866; 1:1000). The following antibodies were purchased from Santa Cruz Biotechnology: Anti-CD95 (1023; 1:500), anti-cyclophilin 40, also known as anti-CYPD (137157; 1:400), anti-His tag (803; 1:500), anti-lamin A (20680; 1:1000), anti-MDM2 (965; 1:500), and anti-ubiquitin (8017; 1:200). Anti-PEPD (Ab86507; 1:500) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374; 1:5000) were purchased from Abcam and EMD Millipore, respectively. Anti-mouse IgG-horseradish peroxidase (IgG-HRP; NA931V; 1:4000–5000) and anti-rabbit IgG-HRP (NA934V; 1:5000) were purchased from GE Healthcare.

Yang L., Li Y., Bhattacharya A, & Zhang Y. (2017). PEPD is a pivotal regulator of p53 tumor suppressor. Nature Communications, 8, 2052.

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