Anti p57

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-p57 is a primary antibody product from Cell Signaling Technology. It is designed to detect the p57 protein, which is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors.

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Lab products found in correlation

6 protocols using anti p57

Quantitative Western Blot Analysis

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Cells protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22μm NC membranes (Sigma) and incubated with specific antibodies. Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad). β-actin antibody was used as control. Anti-E2F1 and anti-p57 were purchased from Cell Signaling Technology, Inc.

Zhang E., Yin D., Han L., He X., Si X., Chen W., Xia R., Xu T., Gu D., De W., Guo R., Xu Z, & Chen J. (2015). E2F1-induced upregulation of long noncoding RNA LINC00668 predicts a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically silencing of CKIs. Oncotarget, 7(17), 23212-23226.

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Western Blot Analysis of Cell Signaling

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Cells were rinsed twice with cold PBS and lysed by RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor cocktail (Roche). Protein (40 μg per sample) was separated by SDS-PAGE using a 10% polyacrylamide gel. The proteins were transferred electrophoretically onto a PVDF membrane. Blotted membranes were blocked in 5% skimmed milk diluted in TBST, followed by incubation with appropriate primary antibodies (anti-EGFR, anti-cyclin D1, CDK4, anti-cyclin D2, anti-p21, anti-p57, anti-MMP-7, anti-MMP-9, anti-cleaved caspase 3, and anti-GADPH; obtained from Cell Signaling Technology and all the antibodies were diluted 1:1000.) overnight at 4°C. The membranes were then washed for 5 minutes for three times with TBST, and subsequently incubated for 1 hour with HRP-linked secondary antibody (Cell Signaling Technology) at room temperature. GADPH was used as an internal control. The blots were detected using an enhanced chemiluminescence kit (Millipore) and subjected to autoradiography using X-ray film.

Zhang T., Cai X., Li Q., Xue P., Chen Z., Dong X, & Xue Y. (2016). Hsa-miR-875-5p exerts tumor suppressor function through down-regulation of EGFR in colorectal carcinoma (CRC). Oncotarget, 7(27), 42225-42240.

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CRISPR Knockout of Cell Cycle Regulators

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p21/p27 dKO and p21/p27/p57 tKO MCF-10A cells were generated according to the manufactural guidance (Integrated DNA technologies). Briefly, predesigned crRNA oligos directed at the locus of CDKN1B and CDKN1C were mixed with tracrRNA oligos tagged fluorescence ATTO 550 in equimolar concentrations to a final duplex concentration of 44 μM and heated at 95 °C for 5 min. Cells were transfected with a gRNA and a Cas9 enzyme. Cells were allowed to recover from the transfection and sorted in single cells by fluorescence-activated cell sorting (FACS). For knockout validation, cells were harvested and immunoblotted by incubating with primary anti-p27 (Cell signaling technology, 3686) and anti-p57 (Cell signaling technology, 2557) and then with anti-rabbit IgG conjugated to horseradish peroxidase (HRP) for chemiluminescent detection.

Kim S., Leong A., Kim M, & Yang H.W. (2022). CDK4/6 initiates Rb inactivation and CDK2 activity coordinates cell-cycle commitment and G1/S transition. Scientific Reports, 12, 16810.

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Immunofluorescence and Immunohistochemistry of Kidney

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Immunofluorescence staining was conducted on frozen or paraffin kidney sections using standard procedures. Briefly, kidney sections were incubated with primary antibodies at 4°C overnight. Primary antibodies used were: anti-p57 (#8298, Cell signaling, Danvers, MA), anti-SIRT1 (#110304, Abcam, Cambridge, MA), anti-nephrin (ab58968, Abcam, Cambridge, MA). After washing with PBS, sections were incubated with Alexa Fluor-488 or Alexa Fluor-568-labeled second antibodies at room temperature for 1 hour. After nuclei were counterstained with 6-diamidino-2-phenylindole, slides were mounted with Aqua PolyMount (Polysciences, Inc., Warrington, PA). Images were acquired using an AxioVision II Microscope with a digital camera (Carl Zeiss, Dublin, CA). For immunohistochemical staining of nitro-tyrosine, deparaffinized kidney sections were incubated with anti-nitrotysine antibody (SC-32757, Santa Cruz Biotechnology Inc., Dallas, TX). After washing, sections were incubated with an anti-rabbit biotinylated secondary antibody at room temperature, and then with the avidin–biotin–peroxidase complex (Vectastain Elite ABC Kit, Vector Laboratories). The reaction products were developed using the 3, 3′-diaminobenzidine substrate and mounted.

Hong Q., Zhang L., Das B., Li Z., Liu B., Cai G., Chen X., Chuang P.Y., He J.C, & Lee K. (2018). Increased podocyte Sirtuin-1 function attenuates diabetic kidney injury. Kidney international, 93(6), 1330-1343.

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Protein Expression Analysis by Western Blot

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Cell protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to 0.22-μm NC membranes (Sigma-Aldrich) and incubated with specific antibodies. Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA), and β-actin antibody was used as the control. Anti-p57 was purchased from Cell Signaling Technology, Inc (Boston, MA, USA). Antibodies against EZH2 and SUZ12 were purchased from Abcam.

Zhang E., He X., Yin D., Han L., Qiu M., Xu T., Xia R., Xu L., Yin R, & De W. (2016). Increased expression of long noncoding RNA TUG1 predicts a poor prognosis of gastric cancer and regulates cell proliferation by epigenetically silencing of p57. Cell Death & Disease, 7(2), e2109-.

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Western Blot Analysis of Cell Signaling

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Western blot was performed as we previously reported [25 (link)]. The antibodies used in this article are listed as follows: anti-p57 (1 : 500, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-p53 (1 : 500, Wanleibio, Xi'an, China), anti-p-p53 (1 : 500, Wanleibio, Shenyang, China), anti-GAPDH (1 : 5000; Genesci, Beijing, China), anti-PCNA (1 : 1000, Boster, Wuhan, China), anti-Cyclin A (1 : 300, Santa Cruz, Dallas, Texas, USA), anti-Cyclin E (1 : 300, Santa Cruz), anti-OCT4 (1 : 500, Santa Cruz), anti-NANOG (1 : 500, PeproTech, Rocky Hill, New Jersey, USA), anti-SOX2 (1 : 1000, Proteintech Group, Rosemont, Illinois, USA), horse-radish peroxidase-conjugated anti-rabbit antibody (1 : 3000, Boster), and anti-mouse antibody (1 : 2000; Boster).

Li N., Du Z., Li Y., Xu W., Yang Y., Peng H., Song T., Qin Q., Lei H, & Hua J. (2021). p57 Suppresses the Pluripotency and Proliferation of Mouse Embryonic Stem Cells by Positively Regulating p53 Activation. Stem Cells International, 2021, 4968649.

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