Luminata crescendo substrate

Manufactured by Merck Group

Sourced in United States

Luminata Crescendo substrate is a chemiluminescent detection reagent used in Western blotting applications. It generates a luminescent signal when reacted with the horseradish peroxidase (HRP) enzyme, which is commonly used to label antibodies in immunoblotting techniques. The substrate's core function is to provide a sensitive and quantitative method for detecting and visualizing target proteins on Western blot membranes.

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Lab products found in correlation

Pvdf membrane, by Merck Group (2 mentions) Bca protein assay, by Thermo Fisher Scientific (2 mentions) Nonfat dry milk, by Cell Signaling Technology (1 mentions) Rabbit anti gfp polyclonal antibody, by Thermo Fisher Scientific (1 mentions) Anti cox7b, by Abcam (1 mentions) Anti gapdh, by Santa Cruz Biotechnology (1 mentions) Anti cox 4, by Cell Signaling Technology (1 mentions) Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Anti β actin, by Merck Group (1 mentions) Bradford method, by Bio-Rad (1 mentions) Anti icam 1, by Santa Cruz Biotechnology (1 mentions) Anti iκb, by Cell Signaling Technology (1 mentions) Protran ba 83, by GE Healthcare (1 mentions) Anti tubulin, by Merck Group (1 mentions) Anti vinculin, by Cell Signaling Technology (1 mentions) Chemidoc xrs system, by Bio-Rad (1 mentions) Mouse anti β actin, by BD (1 mentions) Rabbit anti mcpip1, by GeneTex (1 mentions) Goat anti rabbit igg, by Cell Signaling Technology (1 mentions)

6 protocols using luminata crescendo substrate

Quantitative Protein Analysis via Western Blot

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Cells or embryos (at least 100 for conditions) were lysed in RIPA buffer containing 1X protease inhibitor cocktail (SIGMA). Cleared proteins extracts were quantified by using the Bradford method (Bio-Rad). For WB, protein samples were separated on 15% SDS-PAGE and transferred to PVDF membrane (Millipore). Membranes were blocked in TBS containing 5% Non Fat Dry Milk (Cell Signaling) and incubated with the follows primary antibodies: anti-GFP 1:500 (Abcam, ab13970), anti GFP rabbit polyclonal antibody (Life technologies, a6445), used 1:1000 (for HEK 293T/17 cells experiments), anti-β-Actin 1:500 (Sigma), anti-GAPDH 1:1000 (Santa Cruz Biotechnology), anti-COX-IV 1:500 (Cell Signaling), anti-COX7B 1:500 (Abcam, ab137094).
Proteins of interest were detected with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:3000, GE Healthcare) and rabbit anti-chicken IgY antibody (Millipore) visualized with the Luminata Crescendo substrate (Millipore) or the Super Signal West Femto substrate (Thermo Scientific), according to the manufacturer’s protocol. Western blotting images were acquired using the Chemidoc-lt imaging system (UVP) and band intensity was calculated using ImageJ software.

Indrieri A., Grimaldi C., Zucchelli S., Tammaro R., Gustincich S, & Franco B. (2016). Synthetic long non-coding RNAs [SINEUPs] rescue defective gene expression in vivo. Scientific Reports, 6, 27315.

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Western Blot Analysis of Protein Expression

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Cells were seeded in 35 mm dishes (50000/dish) two days prior to sample collection. Samples were washed once with cold PBS, then lysed in hot buffer (1% (w/v) SDS, 10% (v/v) glycerol, 10% (v/v) β-ME, 40 mM Tris pH 6.8, 0.01% (w/v) bromophenol blue). Proteins were resolved on polyacrylamide gels run under denaturing conditions, then transferred to nitrocellulose membrane (Protran BA-83, GE Healthcare). Membranes were blocked with 5% (w/v) skim milk powder in TBS-T prior to overnight incubation with primary antibody (anti-IκB⍺, Cell Signalling Technology #9242, 1:1,000; anti-ICAM1, Santa Cruz Biotechnology sc-8439, 1:200; anti-⍺-Tubulin, Sigma Aldrich T6199, 1:4,000; anti-Vinculin, Cell Signalling Technology 4650S, 1:1,000). Membranes were washed with TBS-T, incubated with HRP-conjugated secondary antibody (CST #7074 or #7076, 1:1,000), washed and developed using Luminata Crescendo substrate (Millipore WBLUR0500). Signal was detected using Carestream Kodak BioMax MR film (Sigma-Aldrich). Unprocessed images of films are provided in Supplementary Figure S7. Signal was quantified using Fiji (Schindelin et al., 2012 (link)).

Downton P., Bagnall J.S., England H., Spiller D.G., Humphreys N.E., Jackson D.A., Paszek P., White M.R, & Adamson A.D. (2023). Overexpression of IκB⍺ modulates NF-κB activation of inflammatory target gene expression. Frontiers in Molecular Biosciences, 10, 1187187.

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Western Blot Analysis of Cx43 and HIF-1α in PASMCs

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Proteins were extracted from cultured PASMCs with RIPA buffer (containing 1% PMSF) for 30 min on ice, and equal amounts of protein were separated by 10% SDS‐polyacrylamide gels and transferred onto PVDF membranes. Western blots were performed using primary antibodies for Cx43 (Abcam, 1:500), phosphorylation of Cx43 at Ser 368 (Affinity, 1:500), HIF‐1α (Beijing ZSBio, 1:500) or β‐actin (Millipore, 1:1000) and the horseradish peroxidase (HRP)‐coupled goat anti‐mouse secondary antibody (for Cx43, Promega, 1:2000) or HRP‐coupled goat anti‐rabbit secondary antibody (for HIF‐1α and β‐actin, Beijing ZSBio, 1:5000). The chemiluminescence signals were detected with the Luminata™ Crescendo substrate (Millipore, WBLUR0100). Densitometric analysis was conducted with a ChemiDoc XRS+System (Bio‐Rad Co. Ltd. USA).

Han X., Zhang W., Wang Q., Li M., Zhang C., Yang Z., Tan R., Gan L., Zhang L., Lan X., Zhang F., Hong T, & Jiang L. (2021). HIF‐1α promotes the proliferation and migration of pulmonary arterial smooth muscle cells via activation of Cx43. Journal of Cellular and Molecular Medicine, 25(22), 10663-10673.

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Western Blot Analysis of MCPIP1 Protein

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Whole cellular extracts from control, modified, and stimulated cells were prepared using 100 μl of RIPA-lysis buffer (0.25% Na-deoxycholate, 0.5% Nonidet P-40, 0.05% SDS, protease inhibitor cocktail, 2.5 mM EDTA in PBS). Protein concentration was determined using the BCA protein assay (Thermo Fisher Scientific). Equal amounts of protein (20 μg/well) were separated on 12% SDS-PAGE gels and electrotransferred onto PVDF membranes (Millipore) in buffer consisting of 25 mM Tris, 0.2 M glycine, and 20% methanol (100 V, 90 min). Non-specific binding sites were blocked with 5% skim milk in TBST buffer (20 mM Tris, pH 7.5, 0.5 M NaCl, 0.05% Tween 20) for 2 h, followed by overnight incubation with the relevant primary antibody: rabbit anti-MCPIP1 (1:2000, GeneTex) or mouse anti-β-actin (1:10,000, BD Bioscience). Membranes were washed extensively in TBST buffer, and then incubated for 2 h in TBST buffer containing 5% skim milk and horseradish peroxidase (HRP)-conjugated secondary antibodies: goat anti-rabbit IgG (1:3000, Cell Signaling) and goat anti-mouse (1:10,000, BD Bioscience). All incubations were performed at 4°C. Membranes were washed (5 × 5 min) in TBST buffer, and blots were developed using Luminata Crescendo Substrate (Millipore). Membranes were exposed to Kodak Medical X-ray Film and developed.

Dobosz E., Wilamowski M., Lech M., Bugara B., Jura J., Potempa J, & Koziel J. (2016). MCPIP-1, Alias Regnase-1, Controls Epithelial Inflammation by Posttranscriptional Regulation of IL-8 Production. Journal of Innate Immunity, 8(6), 564-578.

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Western Blot Analysis of Apoptosis Regulators

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Whole cell lysates were prepared, resolved and blotted as previously described [5 (link)]. Membranes were probed with the following primary antibodies: anti-PTEN (Cell Signaling, Beverly, MA, USA); anti-c-FLIP (Enzo Life Sciences, Exeter, UK); anti-cIAP-1 (Santa Cruz, Heidelberg, Germany); anti-Bcl-2 (Cell Signaling, Beverly, MA, USA); anti-TNFR-1 (Santa Cruz, Heidelberg, Germany); and anti-Caspase-8 (Millipore, Billerica, MA, USA) at 4°C overnight. Membranes were washed three times with 1X PBS + 0.05% Tween-20 before being incubated with the appropriate horseradish peroxidase (HRP)-tagged secondary antibody (GE Healthcare, UK). Immunolabelled proteins were detected using the Luminata Crescendo substrate (Millipore, Billerica, MA, USA). Membranes were reprobed with GAPDH primary antibody (ABD Serotec, UK) to ensure equal loading.

Armstrong C.W., Maxwell P.J., Ong C.W., Redmond K.M., McCann C., Neisen J., Ward G.A., Chessari G., Johnson C., Crawford N.T., LaBonte M.J., Prise K.M., Robson T., Salto-Tellez M., Longley D.B, & Waugh D.J. (2016). PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy. Oncotarget, 7(7), 7885-7898.

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Western Blot Analysis of Bacterial Proteins

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Bacterial cultures were collected at different time points for preparation of total protein samples. The proteins were separated by SDS-PAGE (8%, 45 min, 220 V). Semidry transfer was performed for 90 min at 20 V using Amersham Protran 0.45 NC Nitrocellulose. Blocked membrane was incubated with primary monoclonal antibodies (Neoclone, 2G10 for σ⁷⁰ and 8RBR for β subunit, 1:1000, 16 h, +4°C, 1% milk PBST). Secondary HRP-conjugated anti-mouse antibodies were used (1:5000, room temperature, 2 h, 1% milk PBST), and the bands were developed using the Luminata crescendo substrate (Millipore) in the chemiluminescence mode in ChemiDoc MP (Bio-Rad).

Pletnev P., Pupov D., Pshanichnaya L., Esyunina D., Petushkov I., Nesterchuk M., Osterman I., Rubtsova M., Mardanov A., Ravin N., Sergiev P., Kulbachinskiy A, & Dontsova O. (2020). Rewiring of growth-dependent transcription regulation by a point mutation in region 1.1 of the housekeeping σ factor. Nucleic Acids Research, 48(19), 10802-10819.

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