Ibright system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The iBright system is a compact and versatile imaging system designed for visualization and analysis of a variety of samples, including gels, blots, and chemiluminescent and fluorescent samples. The system provides high-quality images and supports a range of applications, including Western blotting, protein and DNA gel imaging, and fluorescence detection.

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Lab products found in correlation

11 protocols using ibright system

Western Blot Analysis of m6A Regulators

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Protein was isolated from fibroids and matched myometrium by homogenization in Radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Following isolation, lysate protein concentration was quantified using the Pierce BCA Protein assay kit and 10 μg of protein were separated on a 10% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham). Membranes were blocked in 5% BSA in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 1 h and probed with METTL3 (1:1000; 15073–1-AP; Proteintech), METTL14 (1:1000; 26158–1-AP; Proteintech), RBM15 (1:1000; VIRMA (1:1000; 25712–1-AP; Proteintech), WTAP (1:1000; 10200–1-AP; Proteintech), CBLL1 (1:1000; 21179–1-AP; Proteintech), FTO (1:1000; 27226–1-AP; Proteintech), ALKBH5 (11:1000; 6837–1-AP; Proteintech), and β-actin (1:5000; A5441; Sigma) antibodies at 4 °C overnight. Following washes in TBST, membranes were blocked in secondary HRP-conjugated antibodies (1:10,000) in 5% BSA in TBST for 1 h, washed, and imaged using iBright system (ThermoFisher). Densitometry analysis was performed using Image J. All protein levels were normalized to respective ACTB which served as loading control.

George J.W., Cancino R.A., Miller J.L., Qiu F., Lin Q., Rowley M.J., Chennathukuzhi V.M, & Davis J.S. (2023). Characterization of m6A modifiers and RNA modifications in uterine fibroids. bioRxiv.

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Western Blot Protein Analysis

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For sample preparation, cells were lysed in RIPA buffer (Sigma Aldrich, #R0278) mixed with protease inhibitor cocktail (1:100 dilution, Sigma Aldrich, #118735). Cells were then centrifuged at 14,000 × rpm for 20 min at 4 °C. The supernatants were collected and transferred to a fresh tube. Samples were boiled at 70 °C for 10 min in NuPage LDS sample buffer (Thermo Fischer Scientific, #NP0007) and 1,4-Dithiothreitol (DTT, Sigma Aldrich, #D9779). Proteins were separated on Novex™ 4–12% Tris/Glycine gel (Thermo Fischer Scientific, #XP04200). Gels were then transferred using the iBlot™ 2 Transfer Stacks, nitrocellulose, regular size (Thermo Fischer Scientific, #IB23001). After transfer, membranes were blocked for 1 h in 1 × Tris-buffered saline (Sigma Aldrich, #T5912) with 0.1% Tween 20 (Sigma Aldrich, # P1379) and 5% (wt/vol) BSA (Sigma Aldrich, # A7906) followed by overnight incubation of the respective antibodies at 4 °C. After TBST washing steps, membranes were incubated for 1 h at room temperature with HRP-conjugated anti-mouse secondary antibodies (1:5000 dilution, Thermo Fisher Scientific, ##G21040). Membranes were then developed with the ECL Prime Western Blotting Detection Reagent (GE Healthcare, # RPN2232), signal was measured and band intensities quantified using an iBright system (Thermo Fisher Scientific).

Plotnikov A., Kozer N., Cohen G., Carvalho S., Duberstein S., Almog O., Solmesky L.J., Shurrush K.A., Babaev I., Benjamin S., Gilad S., Kupervaser M., Levin Y., Gershovits M., Ben-Avraham D, & Barr H.M. (2020). PRMT1 inhibition induces differentiation of colon cancer cells. Scientific Reports, 10, 20030.

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Adiponectin Multimer Separation and Analysis

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FPLC was performed as previously described^{40 (link)}. Serum (50 µL) from two representative noncarrier or carrier siblings was injected into an ÄKTA Go FPLC (GE Healthcare). A Superdex 200 10/300 GL column (GE Healthcare) was used to separate adiponectin complexes in HEPES/Ca²⁺ buffer (25 mM HEPES; 150 mM NaCl; and 1 mM CaCl₂, pH 7.4). 250 µL fractions were collected over a 20 mL retention volume. The retention volumes found to contain adiponectin were then used to run Western blots to determine HMW, LMW and trimeric adiponectin. Samples were run on an SDS-Page gel (BioRad Criterion TGX) after being reduced in Laemmli and 355 mM 2-mercaptoethanol and boiled for 10 min. The gel was transferred to PVDF membrane (BioRad). The membranes for each patient were blocked in 5% BSA then probed for adiponectin overnight with rabbit polyclonal anti-adiponectin at a 1:1000 dilution (Abcam, ab75989). There were washed then stained with goat-anti-rabbit AlexaFluor Plus 680 (Invitrogen) at a 1:4000 dilution. Then washed again and imaged on a ThermoFisher iBright system. Western blots derive from the same experiment and were processed in parallel.

Simeone C.A., Wilkerson J.L., Poss A.M., Banks J.A., Varre J.V., Guevara J.L., Hernandez E.J., Gorsi B., Atkinson D.L., Turapov T., Frodsham S.G., Morales J.C., O’Neil K., Moore B., Yandell M., Summers S.A., Krolewski A.S., Holland W.L, & Pezzolesi M.G. (2022). A dominant negative ADIPOQ mutation in a diabetic family with renal disease, hypoadiponectinemia, and hyperceramidemia. NPJ Genomic Medicine, 7, 43.

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Protein Analysis of PFOA Exposure

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For protein analysis, cells were exposed to vehicle control, 1, or 10 μM PFOA for 24 h or 96 h. Cells were then washed in ice cold PBS followed by incubation in RIPA buffer with added protease and phosphatase inhibitor cocktail (ThermoFisher, Waltham, MA) on ice. Lysed cells were centrifuged at 12,000xG for 15 min at 4 °C. Supernatant was collected and protein content was measured using a bicinchoninic acid assay (BCA) per manufacturers protocol (ThermoFisher, Waltham, MA). SDS-PAGE was used to separate the proteins and then transferred to a nitrocellulose membrane. Membranes were blocked in 5% milk in tris-buffered saline with 0.2% Tween-20 (TBSTw) for 1 h followed by incubation in primary antibody (Supplemental Table 1) overnight at 4 °C. Following washes in tris-buffered saline with 0.2% Tween-20 (TBSTw) (3 ×10 min), membranes were incubated with appropriate secondary antibody (Supplemental Table 1) in fresh blocking solution for 1 h at room temperature and subsequently washed with TBSTw (3 ×10 min). Signals were detected using ECL detection substrate and captured on the iBright system (ThermoFisher, Waltham, MA). Densitometry of the appropriately sized bands was measure using ImageJ software (https://imagej.nih.gov). Proteins of interest were normalized to ACTB by dividing protein of interest’s densitometric value by the densitometric value obtained from total ACTB.

Morgan D., Mahe C., Mayanja B, & Whitworth J.A. (2002). Progression to symptomatic disease in people infected with HIV-1 in rural Uganda: prospective cohort study. BMJ : British Medical Journal, 324(7331), 193-197.

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Protein Extraction and Western Blotting

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The cells were lysed with NP-40 buffer (Thermo), and 30 µg of protein was loaded into Mini-PROTEAN precast gels (Bio-Rad, Hercules, CA, USA). Protein transfer was conducted using a Trans-Blot Turbo Transfer System (Bio-Rad). Immunoreactions were detected with SuperSignal™ West Pico or Femto substrates (Thermo) using an iBright System (Thermo). All antibodies were purchased from CST.

Lim B., Yoo D., Chun Y., Go A., Kim J.Y., Lee H.Y., Boohaker R.J., Cho K.J., Ahn S., Lee J.S., Jung D, & Choi G. (2023). Integrative Analyses Reveal the Anticancer Mechanisms and Sensitivity Markers of the Next-Generation Hypomethylating Agent NTX-301. Cancers, 15(6), 1737.

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LPS-Induced Protein Expression Analysis

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After HepG2 cells were treated with 1 μg/mL LPS in serum-free medium, they were exposed to various concentrations of DHP-3 (50-400 µM). Cells were scraped and collected by centrifugation, and cell pellets were lysed with the sample buffer containing 50 mM Tris-HCl (pH 6.8), 2% w/v sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.1% w/v bromophenol blue. Cell lysates were sonicated in an ice bath for 15 sec, heated for 4 min at 100°C, separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane in a Tris/glycine transfer buffer. The membrane was blocked with PVDF Blocking Reagent (Toyobo, Tokyo, Japan), and then incubated with primary antibodies. After washing with phosphate buffered saline-Tween20 (PBS-Tween20), horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG and goat anti-rabbit IgG were applied. The blots were developed using a western blotting detection reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the band intensities were estimated using the VersaDoc™ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and the iBright system (Thermo Fisher Scientific).

Esaki M., Ishida T., Miyauchi Y, & Takechi S. (2020). The effect of dihydropyrazines on lipopolysaccharide-stimulated human hepatoma HepG2 cells via regulating the TLR4-MyD88-mediated NF-κB signaling pathway. The Journal of toxicological sciences, 45(7).

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DHP-3 Attenuates LPS-Induced Cell Stress

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HepG2 cells were exposed to various concentrations of DHP-3 (50-400 µM) after treatment with 1 μg/mL LPS in a serum-free medium. The cells were then lysed with a sample buffer containing 50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.1% bromophenol blue. Cell lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with PVDF Blocking Reagent (TOYOBO Co. Ltd., Tokyo, Japan) and then incubated with primary antibodies. After washing with phosphate-buffered saline with 0.1% Tween20, the membranes were treated with HRP-conjugated secondary antibodies. SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA) was used as the HRP substrate. The signals were detected using the VersaDoc™ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and iBright system (Thermo Fisher Scientific).

Sawai M., Miyauchi Y., Ishida T, & Takechi S. (2022). Dihydropyrazine suppresses TLR4-dependent inflammatory responses by blocking MAPK signaling in human hepatoma HepG2 cells. The Journal of toxicological sciences, 47(9).

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Western Blot Analysis of MRP1 and P-gp

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Western blot assays were carried out following our protocols previously described.31 (link) Briefly, total cell lysates were prepared in RIPA buffer (Beyotime, Shanghai, China). Total proteins were separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking with 5% skim milk for 2 h, the membranes were incubated with the primary antibodies against MRP1 (Cell Signaling Technology, Boston, MA, USA), P-gp (GeneTex, Irvine, CA, USA). Blots then were incubated with the secondary HRP-conjugated antibody (1:5000, Zhongshan Goldenbridge, Beijing, China). Protein bands were visualized using ECL detection reagent and normalized by β-actin (Cell Signaling Technology, Cell Signaling Technology, Boston, MA, USA). Bio-rad Chemidoc^TM XRS⁺ (three Western blots were shown in the supplementary materials. For first blot, exposure time: 0.05 seconds for β-actin, 0.131 seconds for MRP1, 0.321 seconds for p-gP) and Invitrogen iBright system (for second and third blot, exposure time: 0.377 seconds for β-actin exposure time: 1.072 seconds for MRP1) equipments were applied to obtain pictures of chemiluminescence.

Yu H., Tang K., Cai Z., Lin X., Huang Y., Yu T., Zhang Q., Wang Q., Wu L., Yang L., Shan H, & Luo H. (2023). Carbon Dots-Based Nanozyme for Drug-Resistant Lung Cancer Therapy by Encapsulated Doxorubicin/siRNA Cocktail. International Journal of Nanomedicine, 18, 933-948.

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Western Blot Analysis of BACE1, SNAP-25, and Tubulin

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Samples were loaded onto 4–12% Bis-Tris gels using MES-SDS running buffer (Invitrogen), transferred to PVDF membranes, and probed for various proteins using standard WB. The resultant blots were detected by ECL and signals were captured by the iBright system (Invitrogen) or film. The antibodies used to detect specific antigens were: for mouse BACE1, EPR19523 (rabbit, Abcam); for human BACE1, MAB5308, (mouse, Millipore, RRID: AB_95207); for SNAP-25, 610366, (mouse, BD, RRID: AB_397752); and for α-tubulin, T5168 (mouse, Sigma, RRID: AB_477579).

Liu L., Lauro B.M., Ding L., Rovere M., Wolfe M.S, & Selkoe D.J. (2019). Multiple BACE1 Inhibitors Abnormally Increase the BACE1 Protein in Neurons by Prolonging its Half-life. Alzheimer's & dementia : the journal of the Alzheimer's Association, 15(9), 1183-1194.

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Western Blot Analysis of ACE2, NRP1, and TMPRSS2

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Cell lysates were collected with RIPA lysis buffer and quantified using a micro-BCA kit (Thermo Fisher Cat#: 10249133). Samples were boiled for 10 min at 95 °C in 5x Laemmli buffer, separated in an SDS-PAGE gel, and transferred to a polyvinylidene difluoride membrane. Membranes were incubated with 5% non-fat milk in tris-buffered saline buffer (10 mM Tris, pH 8.0, 150 mM NaCl) with 0.1% Tween-20 detergent (TBST) for 1 h and incubated with anti-ACE2 (Bioss Inc Cat #: BS-1004R), anti-NRP1 (Bio-Techne Corporation Cat#: NBP2-67539), anti-TMPRSS2 (Bio-Techne Corporation Cat#: NBP3-00492), anti-Phospho-AMPKα (Thr172) (Cell Signaling Cat#: 2531), and anti-β-actin (Sigma Aldrich Cat#: A5441) primary antibodies at 4 °C for 16 h (dilution 1:1000 or 1:500 for the pull-down assay). Membranes were washed thrice for 10 min and incubated with a secondary anti-rabbit-HRP antibody (Cell signaling Cat#:7074) for 1 h (1:5000 dilution). Blots were washed again with TBST and developed with ECL substrate (Bio-Rad Cat#: 1705061) on an iBright system (Invitrogen). Band densitometry was performed using ImageJ.

Mercado-Gómez M., Prieto-Fernández E., Goikoetxea-Usandizaga N., Vila-Vecilla L., Azkargorta M., Bravo M., Serrano-Maciá M., Egia-Mendikute L., Rodríguez-Agudo R., Lachiondo-Ortega S., Lee S.Y., Eguileor Giné A., Gil-Pitarch C., González-Recio I., Simón J., Petrov P., Jover R., Martínez-Cruz L.A., Ereño-Orbea J., Delgado T.C., Elortza F., Jiménez-Barbero J., Nogueiras R., Prevot V., Palazon A, & Martínez-Chantar M.L. (2022). The spike of SARS-CoV-2 promotes metabolic rewiring in hepatocytes. Communications Biology, 5, 827.

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