Femto maximum sensitivity substrate

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

The Femto Maximum Sensitivity Substrate is a chemiluminescent substrate designed for the detection of very low levels of target proteins in Western blotting applications. It provides a high signal-to-noise ratio to enable the visualization of faint bands, making it suitable for the detection of low-abundance proteins.

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Lab products found in correlation

Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (6 mentions) Image lab software, by Bio-Rad (4 mentions) Supersignal west pico, by Thermo Fisher Scientific (4 mentions) Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Supersignal west pico plus, by Thermo Fisher Scientific (3 mentions) Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Chemidoc xrs imager, by Bio-Rad (3 mentions) β actin, by Merck Group (3 mentions) Chemidoc imaging system, by Bio-Rad (2 mentions) Trans blot turbo system, by Bio-Rad (2 mentions) Chemidoc system, by Bio-Rad (2 mentions) Proteinase k, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Phospho p38, by Merck Group (2 mentions) Image studio lite software, by LI COR (1 mentions) Immobilon p, by Merck Group (1 mentions) Hrp conjugated secondary antibody, by Agilent Technologies (1 mentions) Odyssey imager, by LI COR (1 mentions) Mini protean tgx precast gel, by Bio-Rad (1 mentions)

24 protocols using femto maximum sensitivity substrate

Western Blot Protein Quantification

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The cells were harvested and homogenized in lysis buffer containing 0.5% NP-40 (v/v) and 0.5%Triton X-100 (v/v) in PBS, supplemented with protease and phosphatase inhibitors (A32959, Pierce, Thermo Scientific). Protein concentration was determined using a BCA protein assay kit (Pierce, Thermo Scientific). Equal amounts of total protein were resolved in Mini-PROTEAN TGX Precast gels (Bio-Rad) and transferred to PVDF membrane (immobilon-P, Millipore). 5% BSA (wt/vol) and 0.1% Tween 20 (v/v) in TBS were used to block the membranes followed by incubation with primary antibodies listed in the (Key Resource Table) overnight at 4°C. HRP-conjugated secondary antibodies (DAKO) were used, and HRP signals were developed with Super-Signal West Pico Chemiluminescent substrate or Femto Maximum sensitivity substrate (Thermo Scientific). The blots were imaged with Odyssey imager (Li-COR Biosciences) or Chemi Doc imaging system (Bio-Rad) and quantified with Image Studio Lite Software (Li-COR Biosciences).

Hemanthakumar K.A., Fang S., Anisimov A., Mäyränpää M.I., Mervaala E, & Kivelä R. (2021). Cardiovascular disease risk factors induce mesenchymal features and senescence in mouse cardiac endothelial cells. eLife, 10, e62678.

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Western Blot Analysis of Endothelial Proteins

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Endothelial cells were lysed in modified Radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH 8, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium desoxycholate, 1% Triton X-100, 150 mM NaCl, 1× protease inhibitor cocktail). Proteins were separated by SDS–polyacrylamide gel electrophoresis gradient (4–20%) gel and transferred onto nitrocellulose membranes and incubated overnight at 4°C with primary antibodies. The following primary antibodies were used in this study: PLVAP (DSHB, Cat#MECA-32); PTGS1 (Cell Signaling Technologies, Cat#9896S); TXNIP (Cell Signaling, Cat#71632); FOXP1 (Cell Signaling Technologies Cat#4402S); TFRC (DSHB, Cat#G1/221/12); ZFP36L2 (Cell Signaling, Cat#2119); ACTN1 (Sigma, Cat#A5044); GAPDH (Millipore Sigma, Cat#MAB374); VCL (Millipore Sigma, Cat#V-9131). Secondary antibodies included: Amersham ECL Rabbit IgG HRP-Linked Whole Antibody (Cat#NA934) and Amersham ECL Mouse IgG, HRP-Linked Whole Antibody (Cat#NA931) both from Cytiva. Immuno-complexes were detected by enhanced chemiluminescence with SuperSignal West Pico PLUS (Thermo Fisher Scientific #PI34580) and Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific #PI34096) using ChemiDoc Touch Imaging System (Bio-Rad Laboratories). Quantification of bands by densitometry analysis was performed using ImageLab Software (Bio-Rad Laboratories).

Afshar Y., Ma F., Quach A., Jeong A., Sunshine H.L., Freitas V., Jami-Alahmadi Y., Helaers R., Li X., Pellegrini M., Wohlschlegel J.A., Romanoski C.E., Vikkula M, & Iruela-Arispe M.L. (2023). Transcriptional drifts associated with environmental changes in endothelial cells. eLife, 12, e81370.

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Western Blot Analysis of HIF and Related Proteins

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Cells were washed with PBS and protein was extracted by adding RIPA buffer supplemented with Protease inhibitor (Thermo Scientific #A32955) and phosphatase inhibitor (PhosSTOP Roche 04906845001). Protein concentration was determined using Pierce^TM BCA protein Assay kit (ThermoFisher Scientific #23227). 30 μg of protein samples were boiled at 95 °C for 5 min and loaded into 4–12% Bis-Tris Plus gels (ThermoFisher Scientific #NW04120BOX or ##NW04125BOX) to run in MOPS SDS running buffer (HIF-1α, HIF-2α, HIF-1β, BACH1, NRF2, phospho-STAT3) or MES SDS running buffer (MAFF, MAFG, MAFK). After transfer using Trans-Blot Turbo system (BioRad), membranes were incubated overnight with primary antibodies. After washing three times, secondary antibodies were incubated for 1 hour. Membranes were washed three times again, and protein bands were visualized using SuperSignal^TM West Dura Extended Duration Substrate (ThermoFisher Scientific #34075) or Femto Maximum Sensitivity Substrate (ThermoFisher Scientific #34094). Image Lab 4.0 was used to collect western blot images from Chemidoc.

Moon E.J., Mello S.S., Li C.G., Chi J.T., Thakkar K., Kirkland J.G., Lagory E.L., Lee I.J., Diep A.N., Miao Y., Rafat M., Vilalta M., Castellini L., Krieg A.J., Graves E.E., Attardi L.D, & Giaccia A.J. (2021). The HIF target MAFF promotes tumor invasion and metastasis through IL11 and STAT3 signaling. Nature Communications, 12, 4308.

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Protein Extraction and Western Blot Analysis

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Frozen Neurospora tissue was ground in liquid nitrogen with a mortar and pestle. Total protein was extracted in buffer (50 mM HEPES (pH 7.4), 137 mM NaCl, 10% glycerol v/v, 0.4% NP-40 v/v, and cOmplete Protease Inhibitor Tablet according to instructions for Roche # 11 836 170 001) and processed as described [130 (link)]. Protein concentrations were determined by Bradford Assay (Bio-Rad # 500–0006). For western blots, equal amounts of total protein (30 μg) were loaded per lane into 4% to 12% Bis-Tris Bolt gels (Invitrogen # NW04125/NW04127) or 8% Bis-Tris Bolt gels (Invitrogen # NW00085/NW00087). Western transfer was performed using the Mini Blot Module (Invitrogen # B1000) and BOLT Transfer Buffer (Invitrogen # BT0006) onto an Immobilon-P PVDF membrane (Millipore # IPVH00010). Primary antibodies used for western blotting were anti-CK1a (1:2,000; rabbit raised) [52 (link)], anti-Tubulin alpha (1:5,000; Sigma # T6199), anti-WC-2 (1:5,000; rabbit raised) [131 (link)], and anti-FRH (1:10,000; rabbit raised) [77 (link)]. The secondary antibodies, goat anti-mouse or goat anti-rabbit HRP, were used at 1:5,000 (Bio-Rad # 170–6516, # 170–6515). SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher # 34578) or Femto Maximum Sensitivity Substrate (Thermo # 34095) was used for detection. Immunoblot quantification and normalization were performed in ImageJ.

Kelliher C.M., Stevenson E.L., Loros J.J, & Dunlap J.C. (2023). Nutritional compensation of the circadian clock is a conserved process influenced by gene expression regulation and mRNA stability. PLOS Biology, 21(1), e3001961.

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Protein Extraction and Western Blot Analysis

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Cells were lysed with cold RIPA buffer (50 mM Tris-HCl at pH7.6, 150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, with protease and phosphatase inhibitors). Muscle tissues were lysed with the same buffer using ceramic beads. The concentration of the lysates was measured with the BCA protein assay kit (Thermo Scientific) following the manufacturer's instructions. The samples were loaded to SDS-polyacrylamide gel (Bio-Rad Laboratories), proteins were transferred to nitrocellulose membranes and incubated with primary antibody against MyHC I (BA-D5, Developmental Studies Hybridoma Bank, 1:250), Nfatc1 (YT5381, Immunoway, 1:1,000) or HSC70 (Santa Cruz Biotechnology, 1:5,000). For Prox1, cell lysates and soleus muscle homogenates were first immunoprecipitated with rabbit-anti-human Prox1 (in-house made, 1:1,000) antibody using Protein G beads, transferred to nitrocellulose membranes and incubated with primary antibody for Prox1 (goat-anti-human, AF2727, R&D, 1:1,000). HRP-conjugated anti-mouse or anti-goat secondary antibodies (Dako, 1:5,000) were used and the signal was visualized by the SuperSignal West Pico or Femto Maximum Sensitivity Substrate (Thermo Scientific). Alternatively, anti-rabbit IRDye800 and anti-mouse IRDye680 were used for detection (Licor, 1:10,000).

Kivelä R., Salmela I., Nguyen Y.H., Petrova T.V., Koistinen H.A., Wiener Z, & Alitalo K. (2016). The transcription factor Prox1 is essential for satellite cell differentiation and muscle fibre-type regulation. Nature Communications, 7, 13124.

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Profiling Cytokine Release from Oligodendrocyte Precursor Cells

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WT and TNFR2^−/− OPCs were cultured for 3DIV in 24-well plates, exposed to a cytokine cocktail for 3 h, then switched to a cytokine-free medium. After overnight incubation, the OPC medium containing released factors was collected, spun down at 300× g for 5 min to remove cell debris, and frozen at −80 °C until further analysis. The experiment was run on three biological replicates (three separate OPC primary cultures), each with two technical replicates/condition. The collected OPC medium (500 μL)/sample) was analyzed for cytokine content using the Proteome Profiler Mouse XL Cytokine Array (#ARY028, R&D Systems), following the manufacturer’s protocol. After overnight incubation at 4 °C, membranes were washed and exposed to biotinylated detection antibody followed by incubation with streptavidin-HRP and Femto Maximum Sensitivity Substrate (ThermoScientific). Finally, membranes were imaged with a ChemiDoc system (Bio-Rad) and densitometric quantification of protein expression was performed with HLImage++ software (https://www.wvision.com/QuickSpots.html, accessed on 30 April 2021) after normalization to preloaded controls.

Desu H.L., Illiano P., Choi J.S., Ascona M.C., Gao H., Lee J.K, & Brambilla R. (2021). TNFR2 Signaling Regulates the Immunomodulatory Function of Oligodendrocyte Precursor Cells. Cells, 10(7), 1785.

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Antibody Panel for DNA Repair Assays

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The following antibodies were used for immunoprecipitations and western blotting: anti-Myc tag (Life Technologies Inc., # 46-1155 and Roche Diagnostics GmbH, # 11667203001), anti-V5 (Life Technologies Inc., # 46-1157), anti-Xpress (Life Technologies Inc., # 46-0528), anti-beta-Actin (ACTB, ab8227, Abcam), anti-DNA–PKcs (ab69527, Abcam and sc390849, Santa Cruz Biotechnology, Inc.), anti-KU70 (E5, sc-17789 and M19, sc-1487, Santa Cruz Biotechnology, Inc.), anti-Ku86 (B1, sc-5280 and C-20, sc-1484, Santa Cruz Biotechnology, Inc.), anti-ARTEMIS (Cell Signaling Technology # D708V, Biolegend # 691602). Secondary HRP-conjugated antibodies were from Bio-Rad (goat-anti-mouse IgG-HRP # 170–6516, goat-anti-rabbit IgG-HRP # 170–6515), Santa Cruz (donkey-anti-goat IgG, sc-2033) and from Rockland (Mouse TrueBlot Ultra, # 18-8817-33). Chemiluminescent substrates were from ThermoFisher Scientific (SuperSignal™ West Pico PLUS # 34579 and Femto Maximum Sensitivity Substrate, # 34095). In V(D)J recombination assays Biotin-anti-H-2K^k (BD Biosciences, # 553591) antibody was used. Transfections were performed with Amaxa™ Cell line Nucleofector Kit T, Lonza (CHO derived cell lines), Amaxa™ Cell line Nucleofector Kit V, Lonza (HCT116 derived cell lines).

Niewolik D, & Schwarz K. (2022). Physical ARTEMIS:DNA-PKcs interaction is necessary for V(D)J recombination. Nucleic Acids Research, 50(4), 2096-2110.

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Detection of DNA-RNA Hybrids by Dot Blot

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Cells were washed in PBS and fixed with 1.1% paraformaldehyde in 0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA and 50 mM HEPES pH7 for 30 min. Glycine was added to a final concentration of 0.125 M to quench the reaction. Subsequently, the cells were lysed in 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 0.3% SDS with protease inhibitors and sonicated for 10 cycles. Following proteinase K (2 mg/ml, ThermoFisher, Waltham, MA, USA) treatment for 1 h at 50°C, DNA was isolated using phenol-chloroform extraction. An equal amount of DNA was spotted onto pre-wet nitrocellulose membrane and cross-linked with UVC for 5 min. As a negative control, one half of the samples were also pre-treated with RNaseH (0.03 U/ng DNA, ThermoFisher) for 3 h at 37°C prior to spotting. The membrane was blocked in 5% BSA containing 0.25% Tween-20 and then incubated with S9.6 antibody (Kerafast, Boston, MA, USA, 1:300) overnight. DNA/RNA-hybrids were detected using Femto Maximum Sensitivity Substrate (ThermoFisher) following incubation with peroxidase-conjugated donkey anti-mouse IgG (Jackson Immunoresearch, Cambridgeshire, UK, 1:10000). To ensure equal loading, the membrane was incubated with antibody to single-stranded DNA (Millipore, Saint-Louis, MO, USA, 1:1000) following treatment with 2.5 M HCl for 15 min to denature the DNA. Similarly, detection of ssDNA was performed following exposure to the secondary antibody.

Wohlberedt K., Klusmann I., Derevyanko P.K., Henningsen K., Choo J.A., Manzini V., Magerhans A., Giansanti C., Eischen C.M., Jochemsen A.G, & Dobbelstein M. (2020). Mdm4 supports DNA replication in a p53-independent fashion. Oncogene, 39(25), 4828-4843.

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Quantitative Western Blot Analysis of SHH and VEGF

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Protein lysates were obtained from WJ-MSC monolayers and homogenized in lysis buffer composed of a 1× protease inhibitor mix (Thermo Scientific). Protein concentration was determined (DC™ Protein Assay; BioRad), and a 50-μg protein concentration was loaded for SDS-PAGE and blotted on 0.45-μm pore nitrocellulose membranes. Membranes were blocked and incubated with anti-SHH or vascular endothelial growth factor (VEGF) antibodies. SHH western blots were carried out as previously described [53 (link)] using a 5E1 antibody (Hybridoma supernatant concentrated from Hybridoma Bank; dilution 1/1000). VEGF was detected using rabbit anti-VEGF (Abcam; ab46154; 1/1000). Different positive control samples were used for both proteins (see Results section). Antigens were detected via chemiluminescence using ECL solutions (SuperSignal™ West Pico or Femto Maximum Sensitivity Substrate; Thermo Scientific). Exposed X-ray films (Fujifilm) were analyzed with the Relative Pixel Intensity tool from ImageJ (NIH, USA).

Zavala G., Prieto C.P., Villanueva A.A, & Palma V. (2017). Sonic hedgehog (SHH) signaling improves the angiogenic potential of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC). Stem Cell Research & Therapy, 8, 203.

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EIN3 Immunoblotting in ACC-Treated Seedlings

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Three-day-old dark-grown seedlings were treated with different concentrations of ACC for 2 h. The harvested seedlings were weighed, and 2× SDS sample buffer was added to the seedlings in proportion to their weight. The samples were then homogenized with a pestle. Subsequently, the same amount of total protein extract from each sample was boiled for 3 min and resolved through SDS-PAGE followed by immunoblotting with anti-EIN3 (Agrisera, Cat# As194273, 1:3000 dilution) or anti-Hsc70 (Enzo Life Science, Cat# ADI-SPA-818-F, 1:20000 dilution). Signals were detected with SuperSignal West Pico or Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Cat# 34580, 34095), and band intensities were measured in Image J software.

Park H.L., Seo D.H., Lee H.Y., Bakshi A., Park C., Chien Y.C., Kieber J.J., Binder B.M, & Yoon G.M. (2023). Ethylene-triggered subcellular trafficking of CTR1 enhances the response to ethylene gas. Nature Communications, 14, 365.

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