Supersignal west dura extended duration substrate

Manufactured by Thermo Fisher Scientific

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SuperSignal West Dura Extended Duration Substrate is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It provides an extended duration of light emission, allowing for longer exposure times and increased signal detection.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (62 mentions) Bca protein assay kit, by Thermo Fisher Scientific (46 mentions) Pvdf membrane, by Merck Group (40 mentions) Pvdf membrane, by Bio-Rad (38 mentions) Ripa buffer, by Thermo Fisher Scientific (33 mentions) Protease inhibitor cocktail, by Roche (26 mentions) Nitrocellulose membrane, by Bio-Rad (26 mentions) Trans blot turbo transfer system, by Bio-Rad (23 mentions) Chemidoc imaging system, by Bio-Rad (23 mentions) Chemidoc mp imaging system, by Bio-Rad (21 mentions) Image lab software, by Bio-Rad (20 mentions) β actin, by Merck Group (19 mentions) Ripa buffer, by Merck Group (19 mentions) Chemidoc xrs system, by Bio-Rad (16 mentions) Protease inhibitor cocktail, by Merck Group (16 mentions) Amersham imager 600, by GE Healthcare (15 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (14 mentions) Gapdh, by Cell Signaling Technology (14 mentions) Bca assay, by Thermo Fisher Scientific (14 mentions) Bca protein assay, by Thermo Fisher Scientific (13 mentions)

799 protocols using supersignal west dura extended duration substrate

Histone H3.3 K27M Mutation Analysis

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Histone protein was purified by acid extraction using previously described techniques (Shechter et al., 2007 (link)). Protein was resolved using standard SDS-PAGE techniques and transferred to a 0.45 μm nitrocellulose membrane (GE Healthcare, RPN2020D). 1μg of protein per lane was probed for H3.3 K27M using 1:1000 dilution primary antibody (Millipore, rabbit polyclonal, ABE419) followed by 1:2500 dilution anti-rabbit HRP-conjugated secondary antibody (GE Healthcare Life Sciences, NA934) and detected by chemiluminescence (SuperSignal West Dura Extended Duration Substrate, ThermoFisher Scientific, 34076). This membrane was re-probed for H3K27me3 using 1:1000 dilution primary antibody (Cell Signaling, rabbit monoclonal, 9733) followed by 1:10000 dilution anti-rabbit IRDye800CW-conjugated secondary antibody (LI-COR, 926–32213). On a separate blot, 0.2 μg from the same protein aliquot was probed for total H3 using 1:1000 dilution primary antibody (Abcam, rabbit polyclonal, ab1791) followed by 1:2500 dilution anti-rabbit HRP-conjugated secondary antibody (GE Healthcare Life Sciences, NA934) and detected by chemiluminescence (SuperSignal West Dura Extended Duration Substrate, ThermoFisher Scientific, 34076). All images were developed using the LI-COR Odyssey Fc.

Larson J.D., Kasper L.H., Paugh B.S., Jin H., Wu G., Kwon C.H., Fan Y., Shaw T.I., Silveira A.B., Qu C., Xu R., Zhu X., Zhang J., Russell H.R., Peters J.L., Finkelstein D., Xu B., Lin T., Tinkle C.L., Patay Z., Onar-Thomas A., Pounds S.B., McKinnon P.J., Ellison D.W., Zhang J, & Baker S.J. (2018). Histone H3.3 K27M Accelerates Spontaneous Brainstem Glioma and Drives Restricted Changes in Bivalent Gene Expression. Cancer cell, 35(1), 140-155.e7.

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Validating DUSP6 Knockdown by Western Blot

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To confirm the knockdown of DUSP6, total protein was harvested with RIPA buffer 48 h post siRNA transfection. Proteins (20 μg per sample) were separated by SDS-PAGE via an 8 % Precise protein gel (Life Technologies) and blotted onto a Immobilon-P transfer membrane (Millipore). Detection of DUSP6 was performed using mouse monoclonal antibody (1:1000; Abcam ab54940) and HRP-conjugated rabbit anti-mouse IgG (H+L) secondary antibody (1:5000; Jackson Immunoresearch 315-035-003), and finally visualized with SuperSignal West Dura Extended Duration Substrate (Life Technologies). The antibody was afterwards removed from the membrane with 10 M urea, 45 mM SDS followed by washing steps with 50 % ethanol, 10 % acetic acid. Consecutively, the membrane was re-probed with a mouse monoclonal anti-beta tubulin antibody (1:1000; Abcam ab7792) and the same secondary antibody as before to confirm equal loading amounts of protein lysate.

Wittig-Blaich S., Wittig R., Schmidt S., Lyer S., Bewerunge-Hudler M., Gronert-Sum S., Strobel-Freidekind O., Müller C., List M., Jaskot A., Christiansen H., Hafner M., Schadendorf D., Block I, & Mollenhauer J. (2017). Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma. Oncotarget, 8(14), 23760-23774.

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Western Blot Analysis of TF2a/TF2b Effects

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A2780/CP70 cells were seeded in 60-mm dishes at the density of 1×10⁶ cells/dish, incubated overnight, and treated with TF2a and TF2b (0, 5, 10 and 20 µM) for 24 h. Then the cells were collected with M-PER Mammalian Protein Extraction reagent (Pierce) supplemented with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies, Grand Island, NY, USA). The total protein levels were detected with BCA Protein assay kit (Pierce). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membrane was blocked with 5% skim milk in Tris-buffer saline containing 0.1% Tween-20 for 1 h at room temperature, and then incubated with specific primary antibodies and appropriate secondary antibodies conjugated with horseradish peroxidase. The antigen-antibody complex was visualized with Super Signal West Dura Extended Duration Substrate (Life Technologies) and ChemiDoc™ MP System (Bio-Rad, Hercules, CA, USA). Protein bands were analyzed with ImageJ software and normalized with GAPDH.

Pan H., Wang F., Rankin G.O., Rojanasakul Y., Tu Y, & Chen Y.C. (2017). Inhibitory effect of black tea pigments, theaflavin-3/3′-gallate against cisplatin-resistant ovarian cancer cells by inducing apoptosis and G1 cell cycle arrest. International Journal of Oncology, 51(5), 1508-1520.

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Protein Extraction and Western Blotting

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To obtain protein extracts, cells were washed with chilled PBS and scraped from culture dishes in aqueous lysis buffer (50mM Tris pH7.5, 150mM NaCl, 10mM EDTA pH8.0, 0.2% Sodium Azide, 50mM NaF, 0.5% NP40) containing cOmplete mini protease inhibitor cocktail (Roche, USA 04693159001) and stored at −80°C. Following thawing, they were centrifuged at top speed on a benchtop centrifuge at 4°C for 20 minutes and the supernatant was assayed for protein concentration with Bradford Reagent (Bio-Rad; 500-0006). 30ug of total protein were separated by SDS-PAGE on NuPage gels (Invitrogen, USA) and transferred to Hybond-P PVDF membrane (GE Healthcare, USA). Membranes were probed with specific primary antibodies and antibody-protein complex detected by HRP-conjugated secondary antibodies and SuperSignal West Dura Extended Duration Substrate (Life Technologies USA; 34075).

Pattabiraman D.R., Bierie B., Kober K.I., Thiru P., Krall J., Zill C., Reinhardt F., Tam W.L, & Weinberg R.A. (2016). Activation of PKA leads to mesenchymal-to-epithelial transition and loss of tumor-initiating ability. Science (New York, N.Y.), 351(6277), aad3680.

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Western Blot Analysis of PLP in Larval Brains

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30 brains were dissected from wandering 3^rd instar larvae of the indicated genotypes. Brains were homogenized in 50 μl 1X SDS running buffer (58 mM Tris pH 6.8, 5% glycerol, 1.95% SDS, 1.55 % DTT, ~0.05% Bromophenol Blue) then incubated at ~100°C for 5 min. Samples were run on a 7.5% gel an d transferred to nitrocellulose using the iBlot system (Thermo Fisher Scientific). PLP was detected using a 1:5000 dilution of the N-terminal antibody, followed by horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:1000, Thermo Fisher Scientific). Detection was performed using SuperSignal West Dura Extended Duration Substrate (Life Technologies) and visualized using a ChemiDoc MP Imaging System (Bio-Rad).

Galletta B.J., Ortega J.M., Smith S.L., Fagerstrom C.J., Fear J.M., Mahadevaruju S., Oliver B, & Rusan N.M. (2020). Sperm Head-Tail Linkage Requires Restriction of Pericentriolar Material to the Proximal Centriole End. Developmental cell, 53(1), 86-101.e7.

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TF3 Dose-Dependent Protein Expression

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OVCAR-3 cells were treated for 24 h with various concentrations of TF3 in 60 mm dishes, and then lysed in 100 μl mammalian protein extraction reagent supplemented with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies, Grand Island, NY, USA). The concentration of protein was measured using a BCA Protein assay kit (Thermo, Waltham, MA, USA). Equal amounts of protein were prepared and separated by SDS-PAGE and transferred onto the nitrocellulose membranes with a Mini-Protean 3 system (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% non-fat milk in Tris-buffer saline containing 0.1% Tween-20 for 1 h at room temperature and incubated with indicated primary antibodies overnight at 4ºC followed by horseradish peroxidase-conjugated secondary antibody for 2 h at 37ºC. Detection was performed by SuperSignal West Dura Extended Duration Substrate (Life Technologies) and ChemiDoc™ MP System (Bio-Rad). Protein bands were quantified with the NIH ImageJ software (NIH), normalized by corresponding GAPDH for analysis.

GAO Y., RANKIN G.O., TU Y, & CHEN Y.C. (2015). Theaflavin-3, 3′-digallate decreases human ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1 pathways, not via MAPK pathways. International Journal of Oncology, 48(1), 281-292.

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Western Blot Analysis of MDM Proteins

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Cell lysates (30 μg) of MDMs were subjected to SDS/PAGE, transferred on to nitrocellulose membranes and probed for different proteins using specific primary antibodies against PPARγ, CD36 and β-actin (Santa Cruz Biotechnology), VDAC1 (voltage-dependent anion channel 1) and SDHB (succinate dehydrogenase subunit B; Abcam), and cytochrome c oxidase 1 (Invitrogen). Antibodies against aconitase were provided by Dr. Scott Ballinger. After labelling with appropriate secondary antibodies, proteins were visualized using SuperSignal West Dura Extended Duration substrate (Life Technologies) using the FluorChem M Western blot imaging system (ProteinSimple). Image analysis and densitomery was performed using AlphaView SA software (ProteinSimple). Quantification was only performed on images in which band intensities were below the saturation threshold, as set by the imaging system. Ponceau S staining was performed on all membranes after transfer to ensure transfer efficiency and equal protein loading (results not shown). β-Actin was also used as loading template. Proteins that migrated near the same molecular mass had duplicate membranes probed.

Datta G., Kramer P.A., Johnson M.S., Sawada H., Smythies L.E., Crossman D.K., Chacko B., Ballinger S.W., Westbrook D.G., Mayakonda P., Anantharamaiah G.M., Darley-Usmar V.M, & White C.R. (2015). Bioenergetic programming of macrophages by the apolipoprotein A-I mimetic peptide 4F. The Biochemical journal, 467(3), 517-527.

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Western Blot Analysis of ACK1 Phosphorylation

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Transfected 293T17 cells were treated with cell lysis buffer (Cell Signaling Technologies) with added complete mini protease inhibitor mixture tablets (Roche), phenylmethylsulfonyl fluoride and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich). Lysates were spun at 8000 rpm for 10 min at 4 °C to pellet cell debris, mixed 2:1 with 3X GS sample buffer with β-ME (75 mM Tris (pH 6.8), 3% sodium dodecyl sulfate, 15% glycerol, 8% β-mercaptoethanol, and 0.1% bromphenol blue) and heated at 95 °C for 5 min. Lysates were run on Criterion 4–15% Tris-HCl gradient gels (Bio Rad), transferred to a PVDF membrane, and blocked for 30 min in Tris-buffered saline with Tween (TBST) with 5% BSA. Blots were probed overnight at 4 °C with anti-ACK1 (TNK2) rabbit antibody (Abcam, catalog no. ab65108), anti-ACK1 Phospho Y284 rabbit antibody (Abcam, ab74091), anti-ACK N-terminal antibody (Abcam ab137506), anti-actin mouse antibody (Millipore), followed by anti-rabbit or anti-mouse IgG HRP conjugate secondary antibodies. Blots were developed using Clarity chemiluminescent substrate (BioRad) or SuperSignal West Dura Extended Duration Substrate (Life Technologies) and imaged using a BioRad ChemiDoc MP Imaging System or exposed to x-ray film.

Maxson J.E., Abel M.L., Wang J., Deng X., Reckel S., Luty S.B., Sun H., Gorenstein J., Hughes S., Bottomly D., Wilmot B., McWeeney S.K., Radich J., Hantschel O., Middleton R.E., Gray N.S., Druker B.J, & Tyner J.W. (2015). Identification and characterization of Tyrosine Kinase Non-receptor 2 mutations in leukemia through integration of kinase inhibitor screening and genomic analysis. Cancer research, 76(1), 127-138.

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Western Blot Analysis of Protein Expression

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Cells were treated for 24 h with DMSO (as vehicle), TF1, TF2a, TF2b or TF3 (20 μM) in 60 mm dishes, and then lysed in mammalian protein extraction reagent supplemented with Halt^™ Protease and Phosphatase Inhibitor Single-Use Cocktail (LifeTechnologies, Grand Island, NY, USA). The concentration of protein was measured using a BCA Protein Assay Kit (Thermo, Waltham, MA, USA). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membrane was blocked with 5% non-fat milk in Tris-buffer saline containing 0.1% Tween-20 for 1 h at room temperature and incubated with the indicated primary antibodies overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibody for 2 h at 37°C. Detection was performed by SuperSignal West Dura Extended Duration Substrate (Life technologies) and ChemiDoc^™ MP System (Bio-Rad, Hercules, CA, USA). Protein bands were quantified with NIH ImageJ software (NIH), normalized by corresponding GAPDH for analysis.

GAO Y., RANKIN G.O., TU Y, & CHEN Y.C. (2016). Inhibitory Effects of the Four Main Theaflavin Derivatives Found in Black Tea on Ovarian Cancer Cells. Anticancer research, 36(2), 643-651.

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Zebrafish Eye Protein Expression

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6 dpf zebrafish larvae were anesthetized in Tricaine. Whole larval eyes were dissected and collected in PBS and transferred to urea buffer (65 mM Tris HCl pH 6.75, 8 M urea, 20% glycerine, 5% SDS, 5% β-mercaptoethanol) containing protease inhibitors (cOmplete EDTA-free Protease Inhibitor Cocktail, Roche). Samples were subsequently lysed using a Sonopuls HD 2070 sonicator, and separated on Mini-PROTEAN TGX 4–15% precast polyacrylamide gels (Bio-Rad). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen). Nonspecific antibody binding was inhibited by incubation in PBST (PBS, 0.05% Tween-20) supplemented with 3% skimmed milk powder. Membranes were probed using antibodies against Syntaxin3 (rabbit, 1:2000, Alomone labs, ANR-005), rSec8 (mouse, 1:1000, Enzo, clone 14G1), SNAP25 (rabbit, 1:5000, StressGen Biotechnologies, VAP-SV002), Rab8 (mouse, 1:500, Novus Biologicals, clone 3G1) and β-actin (mouse, 1:1000, Sigma, A1978). HRP-conjugated goat anti-mouse (1:3000, Merck) or anti-rabbit (1:5000, Merck) were used to detect proteins of interest and subsequently visualized by chemiluminescence using luminol/peroxide substrate (SuperSignal West Dura Extended Duration Substrate, Life Technologies) and an ImageQuant LAS 4000 imager.

Ojeda Naharros I., Gesemann M., Mateos J.M., Barmettler G., Forbes A., Ziegler U., Neuhauss S.C, & Bachmann-Gagescu R. (2017). Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors. PLoS Genetics, 13(12), e1007150.

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