Supersignal west dura chemiluminescent substrate

Manufactured by Cell Signaling Technology

SuperSignal West Dura Chemiluminescent Substrate is a laboratory product designed for the detection of proteins in Western blot analysis. It is a chemiluminescent substrate that emits light when combined with a horseradish peroxidase (HRP)-labeled detection system, allowing for the visualization of target proteins.

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Chemiluminescent substrate (7 citations)

6 protocols using supersignal west dura chemiluminescent substrate

Quantification of Cell Cycle Regulators

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PCa cells were cultured in RPMI 1640 with 10% FBS and 1% P/S. Whole cell lysates were prepared from cells, separated on 4% to 20% Tris-Glycine gels and transferred to PVDF membranes. The membranes were incubated with 5% milk for 1 h and incubated with primary antibodies overnight at 4 °C. Primary antibodies used were as follows: p27Kipl (1:1000 dilution, cat. 3686, Cell Signaling), p21 Waf1/Cipl (1:1000 dilution, cat. 2947, Cell Signaling), p16 (1:1000 dilution, cat. 80772, Cell Signaling), PPARγ (1:1000 dilution, cat. 2443, Cell Signaling), p70S6K (1:1000 dilution, cat. 2708, Cell Signaling), phospho-p70S6K (1:1000 dilution, cat. 9234, Cell Signaling). Blots were incubated with peroxidase-coupled anti-rabbit IgG secondary antibody (cat. 7074, 1:2000 dilution, Cell Signaling) for 1 h, and protein expression was detected with SuperSignal West Dura Chemiluminescent Substrate (cat. Prod 34075, Thermo Scientific, Rockford, IL). Membranes were reprobed with monoclonal anti-β-actin antibody (1:1000 dilution, cat. 4970, Cell Signaling) to control for equal loading.

Jung Y., Cackowski F.C., Yumoto K., Decker A.M., Wang Y., Hotchkin M., Lee E., Buttitta L, & Taichman R.S. (2020). Abscisic acid regulates dormancy of prostate cancer disseminated tumor cells in the bone marrow. Neoplasia (New York, N.Y.), 23(1), 102-111.

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ANXA2 and CXCL12 Signaling in PC3 Cells

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PC3 cells (89–90% confluent) were cultured in 6 well plates in RPMI (1ml) without FBS for 18 hours. After serum starvation, the cells were treated with ANXA2 (1μg/ml), CXCL12 (200ng/ml), or combination of ANXA2 (1μg/ml) and CXCL12 (200ng/ml) for 30 min at 37°C. Whole cell lysates were prepared from cells, separated on 10% SDS-polyacrylamide gel and transferred to PVDF membrane. The membranes were first incubated with 5% milk for 1 hour prior to the addition of antibodies to p-AKT (cat. 9271, 1:1,000 dilution, Cell Signaling, Danvers, MA), and AKT (cat. 9272, 1:1,000 dilution, Cell Signaling) followed by overnight at 4°C. Subsequently the blots were incubated with peroxidase-coupled anti-rabbit IgG secondary antibody (cat. 7074, 1:2,000 dilution, Cell Signaling) for 1 hour, and protein expression was detected with SuperSignal West Dura Chemiluminescent Substrate (cat. Prod 34075, Thermo Scientific, Rockford, IL).

Jung Y., Wang J., Lee E., McGee S., Berry J.E., Yumoto K., Dai J., Keller E.T., Shiozawa Y, & Taichman R.S. (2014). Annexin 2-CXCL12 Interactions Regulate Metastatic Cell Targeting and Growth in the Bone Marrow. Molecular cancer research : MCR, 13(1), 197-207.

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Epigenetic Regulation of Neuroendocrine Markers in PCa Cells

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PCa cells were cultured in RPMI 1640 with 10% FBS and 1% P/S. The cells were treated with vehicle or 5-Aza (5µM) for 10 days at 37°C. Whole cell lysates were prepared from cells, separated on 4–20% Tris-Glycine gel and transferred to PVDF membranes. The membranes were incubated with 5% milk for 1 hour and incubated with primary antibodies overnight at 4°C. Primary antibodies used were as follows: N-Myc downstream regulated 1 (NDRG1, 1:1,000 dilution, cat. 9485, Cell Signaling,), Synaptophysin (1:1,000 dilution, cat. 4329, Cell Signaling,), Enolase-2 (ENO2, 1:1,000 dilution, cat. 9536, Cell Signaling,), H3K9me3 (1:1,000 dilution, cat. 13969, Cell Signaling), and H3K27me3 (1:1,000 dilution, cat. 9733, Cell Signaling). Blots were incubated with peroxidase-coupled anti-mouse IgG secondary antibody (cat. 7076, 1:2,000 dilution, Cell Signaling) or peroxidase-coupled anti-rabbit IgG secondary antibody (cat. 7074, 1:2,000 dilution, Cell Signaling) for 1 hour, and protein expression was detected with SuperSignal West Dura Chemiluminescent Substrate (cat. Prod 34075, Thermo Scientific, Rockford, IL). Membranes were reprobed with monoclonal anti-β-Actin antibody (1:1,000 dilution, cat. 4970, Cell Signaling) to control for equal loading.

Lee E., Wang J., Jung Y., Cackowski F.C, & Taichman R.S. (2018). Reduction of Histone Marks, H3K9me3 and H3K27me3 by Epidrug Induces Neuroendocrine Differentiation in Prostate Cancer. Journal of cellular biochemistry, 119(4), 3697-3705.

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Protein Expression Analysis of PCa Cells

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PCa cells were cultured in RPMI 1640 with 10% FBS and 1% P/S. Whole cell lysates were prepared from cells, separated on 4–20% Tris-Glycine gels and transferred to PVDF membranes. The membranes were incubated with 5% milk for 1 hour and incubated with primary antibodies overnight at 4°C. Primary antibodies used were as follows: monoclonal anti-Cyclin A2 (1:1,000 dilution, cat. 4656, Cell Signaling, Danvers, MA), monoclonal anti-CDK2 (1:1,000 dilution, cat. sc-63, Santa Cruz, Santa Cruz, CA), monoclonal anti-p21 (1:1,000 dilution, cat. 2947, Cell Signalng), monoclonal anti-Mer (1:1,000 dilution, cat. 4319, Cell Signaling), p-Mer (1:1000 dilution, cat. ab14921, Abcam). Blots were incubated with peroxidase-coupled anti-rabbit IgG secondary antibody (cat. 7074, 1:2,000 dilution, Cell Signaling) for 1 hour, and protein expression was detected with SuperSignal West Dura Chemiluminescent Substrate (cat. Prod 34075, Thermo Scientific, Rockford, IL). Membranes were reprobed with monoclonal anti-α-Tubulin (1:1,000 dilution, cat. 2125, Cell Signaling) or monoclonal anti-β-actin antibody (1:1,000 dilution; cat. 4970, Cell Signaling) to control for equal loading.

Jung Y., Decker A.M., Wang J., Lee E., Kana L.A., Yumoto K., Cackowski F.C., Rhee J., Carmeliet P., Buttitta L., Morgan T.M, & Taichman R.S. (2016). Endogenous GAS6 and Mer receptor signaling regulate prostate cancer stem cells in bone marrow. Oncotarget, 7(18), 25698-25711.

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Protein Expression Analysis in Cells

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Whole cell lysates were separated on 4-20% Tris-Glycine gel and transferred to PVDF membranes. The membranes were incubated with 5% milk for 1 hour and incubated with primary antibodies overnight at 4°C. Primary antibodies were as follows: anti-EPCAM (cat. 2626, Cell Signaling, Danvers, MA), anti-CK18 (cat. 4548, Cell Signaling), anti-CD24 (cat. ab179821, Abcam, Cambridge, MA), anti-CD44 (cat. 3578, Cell Signaling), anti-CK5 (cat. 25807, Cell Signaling), and anti-Synaptophysin (SYN, cat. 4329, Cell Signaling). Blots were incubated with peroxidase-coupled anti-mouse IgG secondary antibody (cat. 7076, Cell Signaling) or peroxidase-coupled anti-rabbit IgG secondary antibody (cat. 7074, Cell Signaling) for 1 hour, and protein expression was detected with SuperSignal West Dura Chemiluminescent Substrate (cat. Prod 34075, Thermo Scientific, Rockford, IL). Membranes were reprobed with monoclonal anti-β-actin antibody (cat. 4970, Cell Signaling) to control for equal loading.

Jung Y., Kim J.K., Lee E., Cackowski F.C., Decker A.M., Krebsbach P.H, & Taichman R.S. (2020). CXCL12γ Induces Human Prostate and Mammary Gland Development. The Prostate, 80(13), 1145-1156.

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Western Blot Analysis of HIF-1α and VEGFB

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Whole cell lysates were separated on 4–20% Tris-Glycine polyacrylamide gel and transferred to PVDF membranes. The membranes were incubated with 5% bovine milk for 1 hour and incubated with primary antibodies overnight at 4°C (anti-HIF-1a cat. 3716, Cell Signaling, Danvers, MA; anti-VEGFB, cat. 2463, Cell Signaling). Blots were incubated with peroxidase-coupled anti-rabbit IgG secondary antibody (cat. 7074, Cell Signaling) for 1 hour, and protein expression was detected with SuperSignal West Dura Chemiluminescent Substrate (cat. Prod 34075, Thermo Scientific, Rockford, IL). Membranes were restained with monoclonal anti-β-actin antibody (cat. 4970, Cell Signaling) to control for equal loading.

Swanson W.B., Omi M., Zhang Z., Nam H.K., Jung Y., Wang G., Ma P.X., Hatch N.E, & Mishina Y. (2021). Macropore Design of Tissue Engineering Scaffolds Regulates Mesenchymal Stem Cell Differentiation Fate. Biomaterials, 272, 120769.

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