Lumi light plus kit

Manufactured by Roche

Sourced in France, United Kingdom, Germany

The Lumi-light Plus kit is a laboratory equipment product designed for the detection and quantification of proteins in Western blot analysis. The kit utilizes a chemiluminescent substrate to enable the visualization of protein bands on a membrane. The core function of this product is to provide a reliable and sensitive method for the detection of proteins in a research or diagnostic setting.

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Lab products found in correlation

Bca protein assay, by Thermo Fisher Scientific (4 mentions) Proteinase inhibitor cocktail, by Merck Group (3 mentions) Can get signal buffer, by Toyobo (3 mentions) Horseradish peroxidase linked secondary antibody, by Cytiva (2 mentions) Immobilon p, by Merck Group (2 mentions) Phosstop, by Roche (2 mentions) Immobilon p pvdf membrane, by Merck Group (2 mentions) Trans blot turbo transfer system, by Bio-Rad (1 mentions) Anti akt, by Cell Signaling Technology (1 mentions) Anti erk, by Cell Signaling Technology (1 mentions) Anti p akt, by Cell Signaling Technology (1 mentions) Anti p erk, by Cell Signaling Technology (1 mentions) Paris kit, by Thermo Fisher Scientific (1 mentions) Anti β actin, by Merck Group (1 mentions) Anti src, by Cell Signaling Technology (1 mentions) Anti human β actin antibody, by Merck Group (1 mentions) Anti phospho src, by Cell Signaling Technology (1 mentions) Horseradish peroxidase labelled secondary antibodies, by GE Healthcare (1 mentions) Phosphatase inhibitor tablet, by Roche (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions)

Close spelling variants of this product

Lumi-Light

11 protocols using lumi light plus kit

Immunoblotting of Plant Proteins

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Protoplasts were disrupted by addition of hot 2×SDS-PAGE buffer with subsequent heating at 95°C for 5 min. Plant total proteins were extracted by grinding 100 mg A. thaliana leaves in 250 μl of hot 2×SDS-PAGE buffer [84 (link)]. Proteins were separated in 10% or 12% SDS-PAGE gels and transferred onto PVDF Immobilon-P membrane (Millipore). Membranes were then blocked in PBS-Tw 1% buffer (150 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 1% Tween-20) with 5% fat-free milk for 2 hours and incubated with specific primary antibodies raised against CP, RTD or P4 proteins [10 (link), 46 (link)]. The protein/antibody complex was detected by chemiluminescence (Lumi-LightPLUS kit, Roche).
To immunodetect the 3a protein, protein samples were run on a 16% acrylamide Tricine-SDS gel [85 (link)] for 2.5 hours as described above and transferred onto Immobilon-P^SQ membrane (Millipore). Membranes were then blocked in PBS-Tw 0.1% buffer with 1% BSA and incubated with primary antibodies raised against the FLAG epitope (Sigma) or a peptide corresponding to the P3a C-terminal 15 amino acids. The protein/antibody complex was detected by chemiluminescence (Lumi-LightPLUS kit, Roche).

Smirnova E., Firth A.E., Miller W.A., Scheidecker D., Brault V., Reinbold C., Rakotondrafara A.M., Chung B.Y, & Ziegler-Graff V. (2015). Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement. PLoS Pathogens, 11(5), e1004868.

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Immunoblot Analysis of GFP Proteins

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For N. benthamiana, acetone‐precipitated TSP were used. For grapevine, proteins were extracted according to Hurkman and Tanaka (1986). Proteins were heated in denaturing buffer, separated by Tris‐glycine SDS‐PAGE and transferred onto a polyvinylidene difluoride membrane using the TransBlot Turbo transfer system (Bio‐Rad, France). After incubation in blocking buffer (10 mm PBS, 0,1% v/v Tween‐20, 5% w/v skim milk), proteins were sequentially probed with rabbit anti‐GFP IgGs (Sigma‐Aldrich, France) at 0.1 μg/mL in blocking buffer and goat anti‐rabbit IgGs conjugated to horseradish peroxidase (Thermo Fisher Scientific, France) at 0.1 μg/mL in blocking buffer. Immunolabelled proteins were detected by enhanced chemiluminescence (ECL) using the Lumi‐Light^PLUS kit (Roche, France) and the Fusion FX imaging system (Vilber Lourmat GmbH, Germany).

Hemmer C., Djennane S., Ackerer L., Hleibieh K., Marmonier A., Gersch S., Garcia S., Vigne E., Komar V., Perrin M., Gertz C., Belval L., Berthold F., Monsion B., Schmitt‐Keichinger C., Lemaire O., Lorber B., Gutiérrez C., Muyldermans S., Demangeat G, & Ritzenthaler C. (2017). Nanobody‐mediated resistance to Grapevine fanleaf virus in plants. Plant Biotechnology Journal, 16(2), 660-671.

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Protein Expression Analysis in Cancer Cell Lines

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UN-ADC12, UN-ADC18, UN-SCC679, and UN-SCC680 cell lines were seeded at 300 000 cells per well in 6-well plates and allowed to attach overnight. Cells were treated with PBS, sunitinib (at concentrations ranging from 100 nM to 1 μM), DC101 (100 μg/ml), or rat IgG1 antibody (100 μg/ml) for 48 h. Cells were lysed and total proteins were extracted as previously described (Catena et al, 2010 (link)). Twenty μg of total protein from each lysate was boiled at 95°C for 5 min, separated by SDS–PAGE under reduced conditions (5% 2-mercaptoethanol), and transferred onto nitrocellulose membranes. The membranes were subsequently blocked in 5% defatted milk–PBS for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-AKT (1:1000; 9272; Cell Signaling Technology, Beverly, MA, USA), anti-pAKT (1:1000; 9271; Cell Signaling Technology), anti-ERK (1:1000; 9102; Cell Signaling Technology), and anti-pERK (1:1000; 9101; Cell Signaling Technology). Blots were then incubated with a horseradish peroxidase-linked secondary antibody (1:2000; Amersham Pharmacia Biotech, Little Chalfont, UK) and developed by chemiluminescence with Lumilight plus kit (Roche diagnostics, Burgess Hill, UK).

Larrayoz M., Pio R., Pajares M.J., Zudaire I., Ajona D., Casanovas O., Montuenga L.M, & Agorreta J. (2014). Contrasting responses of non-small cell lung cancer to antiangiogenic therapies depend on histological subtype. EMBO Molecular Medicine, 6(4), 539-550.

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Western Blot Analysis of TRAP1 and Apoptosis

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Protein and total RNA were extracted using Paris kit (Ambion-Life Technologies Ltd, Paisley, UK) according to the manufacturer’s instructions. Thirty μg of total protein from each lysate were boiled at 95 ºC for 5 min, separated by SDS/PAGE under reduced conditions (5% 2-mercaptoethanol) and transferred onto a nitrocellulose membrane. The membranes were subsequently blocked in 5% defatted milk-PBS for 1 h and incubated overnight at 4ºC with a primary antibody anti TRAP1 (1:1000, Labvision) or anti β-actin (1:10000, Sigma, Dorset, UK). Blots were then incubated with a horseradish peroxidase-linked secondary antibody (1:5000; Amersham Pharmacia Biotech, Little Chalfont, UK) and developed by chemoluminiscence with Lumilight plus kit (Roche diagnostics, Burgess Hill, UK). Apoptosis detection by western blotting was performed as described before (27 (link)).

Agorreta J., Hu J., Liu D., Delia D., Turley H., Ferguson D.J., Iborra F., Pajares M.J., Larrayoz M., Zudaire I., Pio R., Montuenga L.M., Harris A.L., Gatter K, & Pezzella F. (2014). TRAP1 Regulates Proliferation, Mitochondrial Function and has Prognostic Significance in NSCLC. Molecular cancer research : MCR, 12(5), 660-669.

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Integrin α5 Protein Extraction and Western Blot

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Protein extraction and western blot were carried out as previously described (Ponz-Sarvise et al, 2011 (link)). The primary antibodies (diluted at 1 : 1000) were as follows: anti-human ITGα5 (Sigma), anti phospho-Src (Cell Signaling, Danvers, MA, USA) and anti-Src (Cell Signaling). An anti-human β-actin antibody (Sigma) at 1 : 10 000 dilution was used as a loading control. Horseradish peroxidase-labelled secondary antibodies (GE Healthcare, Waukesha, WI, USA) against the corresponding primary antibodies were used. Immunoreactive bands were visualised by a chemiluminescent method using the Lumi-lightPLUS kit (Roche, Palo Alto, CA, USA).

Larzabal L., de Aberasturi A.L., Redrado M., Rueda P., Rodriguez M.J., Bodegas M.E., Montuenga L.M, & Calvo A. (2014). TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis. British Journal of Cancer, 110(3), 764-774.

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Protein Extraction and Western Blot Analysis

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Cells were harvested from 6-well plates. For the extraction of protein in tissues, excised organs were homogenized in NP-40 lysis buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40) supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor tablet (PhosStop, Roche). The lysate was centrifuged for 10 min at 15000 x g (maximum) at 4 °C, and the supernatant was then collected. The protein concentration was determined by BCA protein assay (Thermo SCIENTIFIC, Waltham, MA) with bovine serum albumin (BSA) standard. Equal amounts of protein samples were loaded, separated by SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (PVDF, Immobilon-P, Millipore). The membranes were blocked in PBS–Tween (0.1%) with 1% BSA or 5% nonfat dried milk and were then probed with the primary antibodies diluted PBST-BSA, 5% nonfat dried milk or Can Get Signal Buffer (TOYOBO, Japan). The bands were detected using Lumi-light Plus kit (Roche) and LAS-3000. Band intensities were quantified with the NIH Image J software. All primary antibodies were used at a dilution of 1:1000.

Ichikawa T., Nakahata S., Fujii M., Iha H, & Morishita K. (2015). Loss of NDRG2 enhanced activation of the NF-κB pathway by PTEN and NIK phosphorylation for ATL and other cancer development. Scientific Reports, 5, 12841.

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Protein Extraction from Tissues

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For protein extraction from tissues, excised organs were homogenized in NP-40 lysis buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 5 mM EDTA, and 1% NP-40) supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitor tablet (Roche, Penzberg, Germany). The lysate was centrifuged for 10 minutes at 15000 × g (maximum) and 4 °C, and the supernatant was then collected. The protein concentration was determined with a BCA protein assay (Thermo Scientific, Waltham, MA, USA), with bovine serum albumin (BSA) as the standard. Equal amounts of protein samples were loaded into SDS-polyacrylamide gels for electrophoresis, and then, the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerrica, MA, USA). The membranes were blocked in PBS–Tween (0.1%) (PBST) with 1% BSA or 5% nonfat dried milk and were then probed with the primary antibodies diluted PBST-BSA, 5% nonfat dried milk or Can Get Signal Buffer (TOYOBO, Osaka, Japan). The bands were detected using a Lumi-light Plus kit (Roche, Penzberg, Germany) and LAS-3000. All primary antibodies were used at a dilution of 1:1000.

Suekane A., Saito Y., Nakahata S., Ichikawa T., Ogoh H., Tsujikawa K, & Morishita K. (2019). CGRP-CRLR/RAMP1 signal is important for stress-induced hematopoiesis. Scientific Reports, 9, 429.

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Western Blot Analysis of Viral Proteins

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CP and RT protein synthesis by virus mutants and wild-type TuYV were analyzed by western blot on agro-infiltrated leaves 5 days after inoculation, using polyclonal antisera raised against TuYV-CP [26 (link)] and TuYV-RT proteins [29 (link)]. Briefly, 100 mg of leaves were sampled and ground in liquid nitrogen. The powder was resuspended in 400 µL of 5X Laemmli buffer [30 (link)]. Boiled samples (15 min at 90 °C) were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and separated proteins were transferred to a nitrocellulose membrane. The membrane was incubated with primary antibodies directed against CP and RT proteins and with a secondary antibody coupled to horseradish peroxidase (goat anti-rabbit-HRP, ThermoFisher). After the addition of horseradish peroxidase substrate (Lumi-LightPLUS kit; Roche, Meylan, France), antibody-antigen complexes were detected with a cooled high sensitivity camera (Syngene Gbox, Fisher Scientific, Illkirch, France).

Boissinot S., Ducousso M., Brault V, & Drucker M. (2022). Bioluminescence Production by Turnip Yellows Virus Infectious Clones: A New Way to Monitor Plant Virus Infection. International Journal of Molecular Sciences, 23(22), 13685.

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Western Blot Analysis of Fatty Tissue Proteome

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Protein fraction extracted from the organic phase of QIAzol Reagent-processed fatty tissue samples (QIAGEN^®, QIAzol™) following the manufacturer’s guidelines, treated with a proteinase inhibitor cocktail (Sigma-Aldrich, Steinheim, Germany) and phosphatase inhibitor tablet (PhosStop™, Roche Diagnostics, IN, USA). Protein specimens were loaded in equal amounts, separated using SDS-poly acrylamide gel (SDS-PAGE) electrophoresis, and then blotted on a polyvinylidene difluoride membrane (Immobilon™-P PVDF membrane, Merck Millipore, MA, USA).The membranes were blocked in PBS–Tween (0.1%) with 1% BSA and were then probed with the diluted primary antibodies (as listed in Table 2). The Lumi-light Plus kit from Roche and the BioRAD chemidoc were used to detect the bands. Intensities of bands were assessed with the NIH Image J software.

Ahmed N., El-Rayes S.M., Khalil W.F., Abdeen A., Abdelkader A., Youssef M., Maher Z.M., Ibrahim A.N., Abdelrahman S.M., Ibrahim S.F., Abdelrahaman D., Alsieni M., Elserafy O.S., Ghamry H.I., Emam H.T, & Shanab O. (2022). Arabic Gum Could Alleviate the Aflatoxin B1-provoked Hepatic Injury in Rat: The Involvement of Oxidative Stress, Inflammatory, and Apoptotic Pathways. Toxins, 14(9), 605.

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Western Blot Analysis of Cell Lysates

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Cell lysate samples were prepared in NP-40 lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (PhosStop, Roche) or by direct lysis in boiling Laemmli SDS sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 25% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue). Protein samples were electrophoresed on 10% SDS-polyacrylamide gel and transferred to polyvinylidine difluoride membranes (Immobilon-P, Millipore). The membranes were blocked in Tris-buffered saline (TBS)-Tween (0.1%) with either 5% bovine serum albumin (BSA) or 5% nonfat dried milk and were then incubated with each primary antibody diluted in TBS containing 0.1% Tween 20 supplemented with either 5% nonfat dried milk or 5% BSA or in the Can Get Signal buffer (TOYOBO). Bound antibody was detected by a Lumi-light Plus kit according to the manufacturer’s instructions (Roche Diagnostics). Band intensities on blots were quantified using NIH Image J software. All primary antibodies were used at a dilution of 1:1,000, except anti-β-actin (1:5,000) and anti-NDRG2 (E20, 1:250). Representative full-gel bots are provided in the Supplementary Fig. 22)

Nakahata S., Ichikawa T., Maneesaay P., Saito Y., Nagai K., Tamura T., Manachai N., Yamakawa N., Hamasaki M., Kitabayashi I., Arai Y., Kanai Y., Taki T., Abe T., Kiyonari H., Shimoda K., Ohshima K., Horii A., Shima H., Taniwaki M., Yamaguchi R, & Morishita K. (2014). Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers. Nature Communications, 5, 3393.

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