Supersignal west dura extended duration

Manufactured by Thermo Fisher Scientific

SuperSignal West Dura Extended Duration is a chemiluminescent substrate used for the detection of Western blots. It provides a longer duration of luminescence compared to standard chemiluminescent substrates, enabling more flexibility in image capture and analysis.

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Lab products found in correlation

Pico plus chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions) A5316, by Merck Group (3 mentions) Mammalian cell lysis kit, by Merck Group (3 mentions) Goat anti mouse rabbit igg, by Jackson ImmunoResearch (3 mentions) Goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions) Ab16502, by Abcam (2 mentions) A5228, by Merck Group (1 mentions) Ab129051, by Abcam (1 mentions) Ab115262, by Abcam (1 mentions) Ab32078, by Abcam (1 mentions) Ab32570, by Abcam (1 mentions) Ab32362, by Abcam (1 mentions) P pdgfrβ, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Ab32199, by Abcam (1 mentions) Sc 53531, by Santa Cruz Biotechnology (1 mentions) Pbs t, by Merck Group (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Anti gapdh, by Merck Group (1 mentions) Odyssey fc imaging system, by LI COR (1 mentions)

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7 protocols using supersignal west dura extended duration

Mammalian Cell Lysis and Western Blot Analysis

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Treated cells or isolated MIVs were lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich), as described previously (Niu et al., 2014 (link)). Equal amounts of protein were electrophoresed in a SDS-polyacrylamide gel under reducing conditions followed by transfer to PVDF membranes. Blots were blocked with 5% BSA in TBS-Tween, and Western blots were probed with antibodies specific for σ-1R (15168-I-AP; Proteintech Group), Src (2108; Cell Signaling), p-PDGFR-β (3161; Cell Signaling), NG2 antibodies (ab129051; Abcam), TBX18 (ab115262; Abcam), αSM (A5228; Sigma-Aldrich), and histone H3 (9715; Cell Signaling) at 1:1,000 dilution; NF-κB (ab16502; Abcam) at 1:2,000 dilution; ganglioside GM1 (bs-2367R; One World Lab) at 1:500 dilution; and p-Src (ab32078; Abcam), PDGFR-β (ab32570; Abcam), Desmin (ab32362; Abcam), and β-actin (A5316; Sigma-Aldrich) at 1:5,000 dilution. Secondary antibodies were alkaline phosphatase conjugated to goat anti-mouse/rabbit IgG, or rabbit anti-goat IgG (1:10,000; Jackson ImmunoResearch Labs). Signals were detected by SuperSignal West Dura Extended Duration or Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). All experiments had at least four biological replicates, and representative blots are presented in the figures.

Niu F., Liao K., Hu G., Sil S., Callen S., Guo M.L., Yang L, & Buch S. (2019). Cocaine-induced release of CXCL10 from pericytes regulates monocyte transmigration into the CNS. The Journal of Cell Biology, 218(2), 700-721.

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Western Blot Analysis of Cellular Proteins

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Brain tissues and treated cells were lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich), as described previously (Liao et al., 2020 (link)). Proteins in equal amounts were electrophoresed in an SDS-polyacrylamide gel under reducing conditions followed by transfer to PVDF membranes. Blots were blocked with 3% BSA in TBS-Tween for 1h, followed by an incubation of antibodies specific hnRNP A2/B1 (1:1000; sc-53531; Santa Cruz), PTEN (1:1000; ab32199; Abcam), p-Akt (1:1000; 9271S; Cell Signaling), Akt (1:1000; 9272S; Cell Signaling), GAPDH (1: 5000, #5174; Cell Signaling) and β-actin (1:5,000; A5316; Sigma-Aldrich). Secondary antibodies were alkaline phosphatase-conjugated to goat anti-mouse/rabbit IgG (1:10,000; Jackson ImmunoResearch Labs). Signals were detected by SuperSignal West Dura Extended Duration or Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). All experiments had at least four biological replicates, and representative blots are presented in the figures.

Liao K., Niu F., Hu G, & Buch S. (2022). Morphine-mediated release of astrocyte-derived extracellular vesicle miR-23a induces loss of pericyte coverage at the blood-brain barrier: Implications for neuroinflammation. Frontiers in Cell and Developmental Biology, 10, 984375.

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Western Blot Analysis of Cochlear Proteins

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Protein samples (30 μg) from mouse cochlear homogenates were separated by SDS-PAGE. After electrophoresis, the proteins were transferred onto a PVDF membrane (Bio-rad, #1620177) and blocked with 5% albumin from bovine serum (Sigma Aldrich, #A7906) in PBS-0.1% Tween-20 (PBS-T) (Sigma Aldrich, #P1379). The membranes were incubated with anti-CaMKKβ (1:1,000, Invitrogen, #PA5–30399), anti-p-CaMKI (T177) (1:1,000, Invitrogen, #PA5–38434), or anti-GAPDH (1:5,000, Millipore, #ABS16) at 4 °C overnight, and then washed three times (10 minutes each) with PBS-T buffer. Membranes were incubated with an appropriate secondary antibody (Cell signaling technology, #7074 or #7076) at a concentration of 1:3,000 for 1 h at RT. Following extensive washing of the membrane, the immunoreactive bands were visualized by SuperSignal West Dura Extended Duration (Thermo Scientific, #34075).
Western blot bands were scanned by LI-COR Odyssey Fc imaging system and analyzed using Image J software. First, the background staining density for each band was subtracted from the band density. Next, the probing protein/GAPDH ratio was calculated from the band densities run on the same gel to normalize for differences in protein loading. Finally, the difference in the ratio of the control and experimental bands was tested for statistical significance.

Wu F., Hill K., Fang Q., He Z., Zheng H., Wang X., Xiong H, & Sha S.H. (2022). Traumatic-noise-induced hair cell death and hearing loss is mediated by activation of CaMKKβ. Cellular and molecular life sciences : CMLS, 79(5), 249.

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Western Blot Analysis of EVs

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Brain tissues, treated cells or EVs were lysed using the Mammalian Cell Lysis kit (Sigma‐Aldrich), as described previously (Liao et al., 2016). Equal amounts of the proteins were electrophoresed in an SDS‐polyacrylamide gel under reducing conditions followed by transfering to PVDF membranes. Blots were blocked with 5% BSA in TBS‐Tween 20. The western blots were then probed with antibodies specific for Iba‐1 (1:1000; 019–19741; Wako), Tsg101 (1:1,000; ab125011; Abcam, Cambridge, MA, USA), Alix (1:1,000, ab117600; Abcam), CD63 (1:1,000; ab216130; Abcam), Flotillin (1:200, Cell Signaling Technology), Calnexin (1:1500; C7617; Sigma‐Aldrich), NF‐κB p65 (1:2,000; ab16502; Abcam), Histone H3 (1:1,000; 9715S; Cell Signaling Technology) and β‐actin (1:5,000; A5316; Sigma‐Aldrich). Secondary antibodies were alkaline phosphatase conjugated to goat anti‐mouse/rabbit IgG (1:10,000; Jackson ImmunoResearch Labs). Signals were detected by SuperSignal West Dura Extended Duration or Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA). All experiments had at least four biological replicates, and representative blots are presented in the figures.

Liao K., Niu F., Hu G., Yang L., Dallon B., Villarreal D, & Buch S. (2020). Morphine‐mediated release of miR‐138 in astrocyte‐derived extracellular vesicles promotes microglial activation. Journal of Extracellular Vesicles, 10(1), e12027.

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Immunoblot Analysis of Signaling Pathways

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Treated cells were lysed in RIPA Buffer supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific) and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Equal amounts of protein were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions followed by transfer to PVDF membranes. Blots were blocked with 5% BSA in TBST and western blots were probed with antibodies specific for CXCR3 (Abcam, ab154845, 1:1000), TLR4 (Novus, NB100-56566, 1:500, Centennial, CO, United States), pTBK1 (Cell Signaling, #5483, 1:1000, Danvers, MA, United States), TBK1 (Abcam, 1:1000), p-IRF3 (Cell Signaling, #4947S, 1:1000), IRF3 (Santa Cruz, sc-33641, 1:500), Histone H3 (Cell Signaling, #9715, 1:1000), Lamin B (Santa Cruz, sc-374015, 1:500), β-actin (Sigma-Aldrich, A5316, 1:5000). Secondary antibodies used were alkaline phosphatase conjugated to goat anti-mouse/rabbit IgG, or rabbit anti-Goat IgG (Jackson ImmunoResearch Labs, 1:10000). Signals were detected by SuperSignal West Dura Extended Duration or Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). Restore PLUS Western Blot Stripping Buffer was used to strip the membrane between two different target protein detection. All experiments had three or four biological replicates with representative blots presented in the figures.

Niu F., Liao K., Hu G., Moidunny S., Roy S, & Buch S. (2021). HIV Tat-Mediated Induction of Monocyte Transmigration Across the Blood–Brain Barrier: Role of Chemokine Receptor CXCR3. Frontiers in Cell and Developmental Biology, 9, 724970.

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Protein Extraction and Western Blot Analysis

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Cells were lysed in a cell lysis buffer (20 mmol/L Tris-HCl pH 7.5, 1% Triton X-102, 0.1% SDS, 150 mmol/L NaCl, 1 mmol/L EDTA, 50 μg/mL Aprotinin and 80 μg/mL PMSF). The cell lysate was subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), followed by electroblotting onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in phosphate-buffered saline (PBS) for 1 h, membranes were probed with specific a mouse monoclonal anti-FLAG (M2; 1:1000; Sigma, St. Louis, MO), a rabbit monoclonal anti-galectin-7 (EPR4287; 1:1000; LifeSpan Biosciences, Inc.), a rabbit polyclonal anti-caspase-3 (#9662; 1:1000; Cell Signaling Technology, Inc., MA), and a rabbit monoclonal anti-beta-actin (#8457(D6A8); 1:1000; Cell Signaling Technology, Inc.) antibodies. Immunoreactive signals were visualized by SuperSignal^® west dura extended duration (Thermo Scientific, Waltham, MA), using Light-capture^® (ATTO, Bioscience and Biotechnology, Tokyo, Japan).

Matsukawa S., Morita K.I., Negishi A., Harada H., Nakajima Y., Shimamoto H., Tomioka H., Tanaka K., Ono M., Yamada T, & Omura K. (2014). Galectin-7 as a potential predictive marker of chemo-and/or radio-therapy resistance in oral squamous cell carcinoma. Cancer Medicine, 3(2), 349-361.

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Mouse Brain Protein Extraction and Analysis

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Protein was extracted from homogenised adult mouse brain (~100 mg brain; 350 μl RIPA buffer containing protease inhibitor cocktail (Roche Diagnostics, Indiana, US)) by centrifugation. Protein yield was determined by Bradford assay (Bio-Rad Laboratories, Hercules, US; bovine serum albumin (BSA) standard). Protein (80 μg) was subjected to gel electrophoresis utilising Criterion XT 1.0 mm gels (Bio-Rad) and transferred to nitrocellulose membrane (Hybond-C Extra 0.45 μm; Amersham Biosciences, Rydalmere, Aus). Membranes blocked in 3% BSA-TBS/T (Tris buffered saline (TBS)/0.1% Tween-20 (T)) overnight (4 °C) were probed for SGTA using ‘full length SGTA’ (ProteinTech), ‘C-terminal SGTA’ (Aviva BioSciences, San Diego, US) or ‘N-terminal SGTA’ (Santa Cruz) antibodies diluted at 1:1,000 (full, C-terminal) or 1:500 (N-terminal) in 1% BSA-TBS/T (60 min, room temperature). Immunoreactivity was detected using polyclonal goat anti-rabbit (1:1,000) or rabbit anti-mouse IgG/HRP (1:500) antibodies (Dako) followed by chemiluminescent visualisation (Super Signal West Dura Extended Duration, Thermo Fisher Scientific, Scoresby, Aus) and ChemiDoc MP imaging (Bio-Rad). Reference proteins used were actin (Santa Cruz Biotechnology, sc-1616, 1:2,000) or Histone H-3 (Abcam, ab7766, 1:500).

Philp L.K., Day T.K., Butler M.S., Laven-Law G., Jindal S., Hickey T.E., Scher H.I., Butler L.M, & Tilley W.D. (2016). Small Glutamine-Rich Tetratricopeptide Repeat-Containing Protein Alpha (SGTA) Ablation Limits Offspring Viability and Growth in Mice. Scientific Reports, 6, 28950.

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