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Azure c500 machine

Manufactured by Azure Biosystems
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The Azure C500 is a compact, automated Western blot imaging system designed for straightforward and efficient protein detection and quantification. It features a high-resolution CCD camera and LED-based illumination to capture clear, high-quality images of chemiluminescent and fluorescent Western blots.

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Lab products found in correlation

β actin, by Santa Cruz Biotechnology (6 mentions) Tris glycine gel, by Thermo Fisher Scientific (5 mentions) Myd88, by Abcam (2 mentions) Epac1, by Abcam (2 mentions) Traf6, by Abcam (2 mentions) Chemilluminescence reagent kit, by Thermo Fisher Scientific (2 mentions) Phosphorylated insulin receptor substrate 1, by Cell Signaling Technology (2 mentions) Total irs 1, by Cell Signaling Technology (2 mentions) Chemiluminescence reagent kit, by Thermo Fisher Scientific (1 mentions) Occludin, by Abcam (1 mentions) Cleaved caspase 1, by Thermo Fisher Scientific (1 mentions) Anti sfpq, by Santa Cruz Biotechnology (1 mentions) Tnf α, by Abcam (1 mentions) Il 1β, by Abcam (1 mentions)

7 protocols using azure c500 machine

1

Western Blot Analysis of Protein Expression

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Cell culture lysates were collected into lysis buffer with both protease and phosphatase inhibitors. Equal amounts of protein were separated using pre-cast tris–glycine gels (Invitrogen, Carlsbad, CA), and blotted onto nitrocellulose membranes. After blocking in TBST (10 mM Tris–HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes were treated with an Epac1, TLR4, MyD88, IRAK, TRAF6, TRAM1, IRF3, occludin, and ZO-1 (Abcam, Cambridge, MA) and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies tagged with horseradish peroxidase. Antigen–antibody complexes were visualized with an Azure C500 machine (Azure Biosystems, Dublin, CA) after application of a chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Western blot band densities were measured using Image Studio Lite software.
Liu L., Jiang Y, & Steinle J.J. (2021). Forskolin regulates retinal endothelial cell permeability through TLR4 actions in vitro. Molecular and cellular biochemistry, 476(12), 4487-4492.
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2

Protein Expression Analysis in Whole Retinal Lysates

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Whole retinal lysates from the mice were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated using pre-cast tris-glycine gels (Invitrogen, Carlsbad, CA), and blotted onto nitrocellulose membranes. After blocking in TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes were treated with a TLR4, IL-1B, TNFa, VEGF, and ICAM1 (Abcam, Cambridge, MA), and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were visualized using a chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) and data was acquired with an Azure C500 machine (Azure Biosystems, Dublin, CA). Western blot band densities were measured using Image Studio Lite software.
Seidel A., Liu L., Jiang Y, & Steinle J.J. (2021). Loss of TLR4 in endothelial cells but not Müller cells protects the diabetic retina. Experimental eye research, 206, 108557.
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3

Insulin Signaling Pathway Analysis

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Whole retinal lysates were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were placed into pre-cast trisglycine gels (Invitrogen, Carlsbad, CA), and blotted onto nitrocellulose membranes. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) with 5% BSA, membranes were treated with a phosphorylated insulin receptor (Tyr 1150/1151), insulin receptor, phosphorylated Akt (Ser473), total Akt, phosphorylated insulin receptor substrate 1 (Ser307), total IRS-1 (Cell Signaling Technology, Danvers, MA) TNF, (Abcam, Cambridge, MA), and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies overnight. The following day, membranes were incubated with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were visualized using Chemiluminescence (Thermo Scientific, Pittsburgh, PA). Data was analyzed on an Azure C500 machine (Azure Biosystems, Dublin, CA). Western blot band densities were measured using Image Studio Lite software.
Jiang Y., Liu L., Li H., Wang J.M, & Steinle J.J. (2020). Insulin Signal Transduction is Impaired in the Type 2 Diabetic Retina. Journal of diabetes and clinical research, 2(1), 12-15.
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4

Western Blotting and IL-1β ELISA for Inflammasome Signaling

Cited 1 time
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Western blotting was performed as previously described [16 (link)]. The primary antibodies used were Epac1 (Ab124162, 1:1000), PKA (Ab75991, 1:500), NLRP3 (Ab263899, 1:500), ASC1 (Ab70627, 1:600), Nek7 (Ab133514, 1:500), P2X7 receptor (Ab109054, 1:500; Abcam, Cambridge, MA), cleaved caspase 1 (Asp297, ThermoFisher PA5–77886, 1:200), and beta-actin (Santa Cruz). The primary antibodies were incubated overnight. Secondary antibodies were conjugated to horseradish peroxide (HRP; Promega, Madison, WI). Bands were visualized using an Azure C500 machine (Azure, Dublin, CA). IL-1β ELISA. IL-1β ELISA (R&D Systems, Menomomie, WI) was performed according to the manufacturer’s instructions, with the exception that the ELISA was run overnight at 4 °C.
Liu L., Jiang Y, & Steinle J.J. (2022). Epac1 and PKA agonists inhibit ROS to reduce NLRP3 inflammasome proteins in retinal endothelial cells. Molecular Vision, 28, 500-506.
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5

Western Blot Analysis of Nuclear Proteins

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Liver nuclei were prepared by centrifugation through 2M sucrose cushions as described [33 (link)] and liver nuclear extracts prepared. About 20 μg of nuclear extract was separated on 7% SDS-polyacrylamide gels, transferred to BA83 nitrocellulose membranes and Western blot performed. Antibodies used were anti-RNA polymerase II (ab817, Abcam, Cambridge, UK), anti-Sfpq (kind gift from Steven Brown, Zurich), and anti-Csnk1δ (sc-55553, Santa Cruz Biotechnology, Dallas, TX). Specific antibody:antigen complexes were detected with matching HRP-conjugated secondary antibodies and Western Bright Sirius enhanced chemiluminescence (Advansta, Mento Park, CA) using an Azure C500 machine (Azure Biosystems, Dublin, CA) and increasing exposure times of 30 sec each. All images were analysed using ImageJ (http://imagej.nih.gov/ij/index.html).
Fonseca Costa S.S., Wegmann D, & Ripperger J.A. (2017). Normalisation against Circadian and Age-Related Disturbances Enables Robust Detection of Gene Expression Changes in Liver of Aged Mice. PLoS ONE, 12(1), e0169615.
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6

Insulin Signaling Pathway Analysis

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Whole retinal lysates were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were placed into pre-cast trisglycine gels (Invitrogen, Carlsbad, CA), and blotted onto nitrocellulose membranes. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) with 5% BSA, membranes were treated with a phosphorylated insulin receptor (Tyr 1150/1151), insulin receptor, phosphorylated Akt (Ser473), total Akt, phosphorylated insulin receptor substrate 1 (Ser307), total IRS-1 (Cell Signaling Technology, Danvers, MA) TNF, (Abcam, Cambridge, MA), and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies overnight. The following day, membranes were incubated with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were visualized using Chemiluminescence (Thermo Scientific, Pittsburgh, PA). Data was analyzed on an Azure C500 machine (Azure Biosystems, Dublin, CA). Western blot band densities were measured using Image Studio Lite software.
Jiang Y., Liu L., Li H., Wang J.M, & Steinle J.J. (2020). Insulin Signal Transduction is Impaired in the Type 2 Diabetic Retina. Journal of diabetes and clinical research, 2(1), 12-15.
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7

Western Blot Analysis of Retinal Signaling

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Whole retinal lysates or cell culture lysates were collected into lysis buffer containing protease and phosphatase inhibitors. For mouse samples, 2 retinas from 1 mouse were pooled together from 5 mice in each group. Equal amounts of protein were separated using pre-cast tris-glycine gels (Invitrogen, Carlsbad, CA), and blotted onto nitrocellulose membranes. After blocking in TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes were treated with a Epac1, TLR4, MyD88, IL-1β, TNFα, IRAK, TRAF6, IRF3, phosphorylated and total NFkB antibodies (Abcam, Cambridge, MA), and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were visualized using a chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) and data was acquired with an Azure C500 machine (Azure Biosystems, Dublin, CA). Western blot band densities were measured using Image Studio Lite software.
Liu L., Jiang Y, & Steinle J. (2021). Epac1 regulates TLR4 signaling in the diabetic retinal vasculature. Cytokine, 144, 155576.
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