Azure imager c300

Manufactured by Azure Biosystems

Sourced in United States

The Azure Imager c300 is a compact and versatile imaging system designed for a wide range of applications in life science research. It offers high-quality image capture and analysis capabilities for various sample types, including gels, blots, and microplates.

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Lab products found in correlation

Nitrocellulose membrane, by Thermo Fisher Scientific (5 mentions) Protease and phosphatase inhibitor cocktail, by Merck Group (4 mentions) Radioimmunoprecipitation assay buffer, by Merck Group (4 mentions) 4 to 12 bis tris protein gradient gels, by Thermo Fisher Scientific (2 mentions) Tnfr1, by Cell Signaling Technology (2 mentions) Il 1r, by Thermo Fisher Scientific (2 mentions) Bis tris gradient gel, by Thermo Fisher Scientific (2 mentions) P nf κb, by Cell Signaling Technology (2 mentions) Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (2 mentions) Nf κb, by Cell Signaling Technology (2 mentions) β actin, by Merck Group (2 mentions) Hrp conjugated secondary antibody, by GE Healthcare (1 mentions) Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions) Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Clarity max western ecl substrate, by Bio-Rad (1 mentions) Ab206996, by Abcam (1 mentions) Pierce bca assay kit, by Thermo Fisher Scientific (1 mentions) Radiance plus, by Azure Biosystems (1 mentions)

Close spelling variants of this product

Azure c300 digital imager system Azure C300 Gel Imager Azure C300 C300 imager

11 protocols using azure imager c300

Simvastatin Modulates Leiomyoma Stem Cell Signaling

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Leiomyoma stem cells were treated with simvastatin (0.01, 0.1, 1 µM) or DMSO (vehicle control) for 48 h. Cells were lysed in a lysis buffer (radioimmunoprecipitation assay buffer, Sigma‐Aldrich) containing a protease and phosphatase inhibitor cocktail (Sigma‐Aldrich). An equal amount of protein lysates was loaded on 4 to 12% Bis‐Tris protein gradient gels (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (Thermo Fisher Scientific) using the mini trans‐blot transfer system (Thermo Fisher Scientific). To detect specific antigens, blots were probed with primary antibodies (Table S2) on a shaker at 4° C overnight, followed by 1 h of room temperature incubation with HRP conjugated secondary antibodies (GE Healthcare). Chemiluminescent Western blot signal was captured was using an Azure Imager c300 (Azure Biosystems). Band signals were quantified with the NIH ImageJ software (version 1.52r).

Afrin S., Ali M., El Sabeh M., Yang Q., Al‐Hendy A, & Borahay M.A. (2022). Simvastatin inhibits stem cell proliferation in human leiomyoma via TGF‐β3 and Wnt/β‐Catenin pathways. Journal of Cellular and Molecular Medicine, 26(5), 1684-1698.

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Tissue Protein Extraction and Western Blot Analysis

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Heart, kidney, pancreas, and skeletal muscle were isolated from 6-month old mice and snap frozen at −80 °C. Protein extraction was carried out using T-PER^™ Tissue Protein Extraction Reagent (ThermoFisher Scientific, Waltham MA) with Halt^™ Protease Inhibitor Cocktail (ThermoFisher Scientific, Waltham MA) and Halt^™ Phosphatase Inhibitor Cocktail (ThermoFisher Scientific, Waltham MA). Protein concentration of each sample was determined by BCA Protein Assay Reagent (Pierce, Rockford IL). Standard electrophoresis was performed. Blocking and antibodies were prepared in filtered 5% BSA. The blots were developed using Clarity Max Western ECL Substrate (Bio-Rad, Hercules CA) and were scanned using Azure Imager C300 (Azure Biosystems, Dublin CA). The densitometric analysis was conducted to quantify the Western blot immunoreactivity using Image J. The primary antibodies used were IGF-I Receptor β (1:1000, #9750S), Phospho-IGF-I Receptor β (1:1000, #3918S), Akt (pan) (1:1000, #4691S), Phospho-Akt (1:2000, #4060S), and Actin (pan) (1:1000, #4968S). All primary antibodies were obtained from Cell Signaling Technology (Danvers MA). The secondary antibody used was goat anti-rabbit IgG-HRP (1:4000; Santa Cruz Biotechnology, Santa Cruz CA).

Dou Y., Darvas M., Sharma K., Mathieu J., Morton J., Tan H., Soto-Palma C., Angelini L.A., McGowan S.J., Niedernhofer L.J., Suh Y., Robbins P.D., Barzilai N, & Ladiges W.C. (2020). Development of an IGF1R longevity variant mouse line using CRISPR/Cas9 genome editing. Trends in biomedical research, 3(1), 121.

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Western Blot Protocol for Protein Analysis

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Cells were lysed in RIPA buffer (Abcam, ab206996) and equal amounts of proteins (quantified with Pierce BCA assay kit, Thermo Fisher, 23252) were separated on 4–15% gradient Criterion precast gels (Bio-Rad 567–1084). Proteins were then transferred onto nitrocellulose membranes (Bio-Rad). Western Blots were developed with chemiluminescence HRP substrate (Radiance plus, Azure Biosystems AC2103) on a digital image analyzer, Azure Imager c300. Uncropped western blots are shown in Figure S9.

Ting A.Y., Kain K.H., Klemke R.L, & Tsien R.Y. (2001). Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proceedings of the National Academy of Sciences of the United States of America, 98(26), 15003-15008.

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Protein Analysis of Myometrium and Leiomyoma Cell Co-culture

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After coculture, the primary and immortalized myometrium and leiomyoma cells and the ELT3 cells were harvested and lysed in a lysis buffer (radioimmunoprecipitation assay buffer; MilliporeSigma) containing a protease and phosphatase inhibitor cocktail (MilliporeSigma). We resolved the same amounts of protein lysates with 4–12% Bis-Tris gradient gels (Thermo Fisher Scientific) and transferred them onto nitrocellulose membranes (Thermo Fisher Scientific). The membranes were blocked by soaking in 5% nonfat milk in Tris-buffered Saline with 0.1% Tween-20 (TBST; Thermo Fisher Scientific) for 1 h at room temperature, and then incubated overnight with antibodies targeting IL-1R (Thermo Fisher Scientific; PA5–21776), TNF-R1 (Cell Signaling Technology; #3736S), toll-like receptor 4 (TLR4; Thermo Fisher Scientific; PA5–26689), PCNA (Cell Signaling Technology; #13110), p NF-κB (Cell Signaling Technology; #3033), NF-κB (Cell Signaling Technology; #8242), or β-actin (MilliporeSigma; A3854) at 4°C on a rocker. After washing with TBST, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature, after which they were visualized using an Azure Imager c300 (Azure Biosystems, Dublin, California, USA). The NIH ImageJ software (version 1.52r) was used to quantitate the protein band signals [28 (link)].

Afrin S., El Sabah M., Manzoor A., Miyashita-Ishiwata M., Reschke L, & Borahay M.A. (2022). Adipocyte coculture induces a pro-inflammatory, fibrotic, angiogenic, and proliferative microenvironment in uterine leiomyoma cells. Biochimica et biophysica acta. Molecular basis of disease, 1869(1), 166564.

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Protein Analysis of Myometrium and Leiomyoma Cell Co-culture

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Western Blot Analysis of Statin and E2 Effects

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Cells were harvested after treatment with simvastatin (0.001, 0.01, 0.1, and 1 μM) and E₂ (10 nM), alone or in combination, at the specified time points. Cells were lysed in a lysis buffer (radioimmunoprecipitation assay buffer, Sigma-Aldrich) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich). An equal amount of protein lysates was resolved using 4 to 12% Bis-Tris protein gradient gels (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (Thermo Fisher Scientific). Membranes were blocked using 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 (TBST, Thermo Fisher Scientific) for 1 h at room temperature and incubated with PCNA (CST, #13110), anti- ER-α (Invitrogen, PA5–16440), pERK1/2 (CST, #4695S), ERK1/2 (CST, #4370), pAKT (Santa Cruz; sc-7985), AKT (Santa Cruz; sc-8312), anti-collagen 1 (Thermo Fisher Scientific, PA5–29569), anti-β-actin (Sigma, A3854), anti-Na,K-ATPase (CST, 23565), or anti-lamin B1 (Invitrogen, PA5–19468) at 4 °C overnight on a rocker. Membranes were washed with TBST, co-incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare, Chicago, IL) for 1 h at room temperature and visualized using an Azure Imager c300 (Azure Biosystems, Dublin, CA). Band signals were quantified using the NIH ImageJ software (version 1.52r, USA).

Afrin S., El Sabeh M., Islam M.S., Miyashita-Ishiwata M., Malik M., Catherino W.H., Akimzhanov A.M., Boehning D., Yang Q., Al-Hendy A., Segars J.H, & Borahay M.A. (2021). Simvastatin modulates estrogen signaling in uterine leiomyoma via regulating receptor palmitoylation, trafficking and degradation. Pharmacological research, 172, 105856.

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Western Blot Analysis of MAPK Phosphorylation

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Proteins were separated by 15% SDS-PAGE and transferred to 0.2 μm PVDF membranes (Bio-Rad, CAD) for 1 h at 0.4 V cm -1 in a Mini-PROTEAN® Tetra Cell (Bio-Rad, CAD). His-tagged proteins were detected using monoclonal anti-polyHis-HRP conjugate (Sigma-Aldrich, CAD). Phosphorylated mitogen-activated protein kinases (MAPK) were detected using phospho-p44/p42 MAPK primary antibody (Cell Signally Technology, Leiden, Netherland), followed by a goat anti-rabbit secondary antibody HRP conjugate (Sigma-Aldrich, CAD) following the manufacturer´s recommendations. Membranes were blocked with 5% skim-milk and HPR activity was detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, CAD) with an Azure imager C300 (Azure Biosystems, CAD), to detect the chemiluminiscent signal.

Muirhead K., & Pérez‐López E. (2020). Plasmodiophora brassicae chitin-binding effectors guard and mask spores during infection.

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Multiplex Cytokine and EMAP II Analysis

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The human pro-inflammatory cytokines IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL_12p70, IL-13, and TNF-α were measured in 150 μL of neat lung lavage fluids by using electrochemiluminescent sulfo-tag labels as implemented in the multi-spot plates in Proinflammatory Human Cytokines Panel I V-plex kits (Meso Scale Discovery). Analyses were done using MESO QuickPlex SQ 120 and Discovery Workbench software (MSD). The expression of Endothelial-Monocyte Activating Polypeptide II (EMAP II) was assessed by Western blot analyses in mouse lung tissue homogenates. Briefly, lung tissues were homogenized in T-PER solution (Thermo Scientific) supplemented with protease inhibitors (cOmplete Protease Inhibitor Cocktail; Sigma). The tissue lysates were analyzed by SDS-gel electrophoresis using AnykD TGX pre-cast gels (Bio-Rad). The following antibodies were used: rabbit polyclonal anti-EMAP II serum (lot D88) (86 (link)) or anti-AIMP1/EMAPII/SCYE1 (Bethyl laboratories # A304-896A-M), rabbit anti-Vinculin (Abcam #ab129002), along with HRP-conjugated goat anti-rabbit secondary antibodies. We imaged the membranes by incubation in Super-Signal West-Femto Substrate (Thermo Fisher) for five minutes, followed by a cumulative 3-min exposure time in Azure C300 imager (Azure Biosystems). Densitometry analyses were done using FIJI software (87 (link)).

Rodriguez-Irizarry V.J., Schneider A.C., Ahle D., Smith J.M., Suarez-Martinez E.B., Salazar E.A., McDaniel Mims B., Rasha F., Moussa H., Moustaïd-Moussa N., Pruitt K., Fonseca M., Henriquez M., Clauss M.A., Grisham M.B, & Almodovar S. (2022). Mice with humanized immune system as novel models to study HIV-associated pulmonary hypertension. Frontiers in Immunology, 13, 936164.

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Quantitative Western Blot Analysis

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Aliquots containing 20 μg of total
proteins from each sample including molecular weight markers, iBright
Prestained Protein Ladder ranging from 11 to 250 kDa, were loaded
in different wells of precast Criterion XT 12% Bis-Tris (1 mm, 18-well)
gels. Gels were run at 200 V for 1 h and transferred onto the nitrocellulose
membrane (Thermo Fisher) at 0.07 A for 17 h. Membranes were then washed
with PBST and cut at the 55 kDa ladder marker for separate antibody
incubations for haptoglobin and gelsolin. The membranes were then
washed in Bio-Rad Blotting-Grade Blocker and separately incubated
in anti-haptoglobin (ab131236) and anti-gelsolin (ab75832) at 1:10000
and 1:25000 dilutions, respectively. Membranes were washed again and
incubated in anti-rabbit IgG-HRP (NA934V) antibodies at 1:3000 dilutions.
Membranes were revealed using an Azure c300 imager (Azure Biosystems,
CA, USA) and image-analyzed using open-access software (GelQuant.NET)
with fluorescence settings at 10 s of absorbance.

Alayi T.D., Tawalbeh S.M., Ogundele M., Smith H.R., Samsel A.M., Barbieri M.L, & Hathout Y. (2020). Tandem Mass Tag-Based Serum Proteome Profiling for Biomarker Discovery in Young Duchenne Muscular Dystrophy Boys. ACS Omega, 5(41), 26504-26517.

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Western Blotting for Inflammasome Activation

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BMDMs were activated with LPS and nigericin as described above and were lysed in situ with 50 μL of 4× NuPAGE LDS sample buffer (Fisher Scientific, Hampton, NH) + 5% β-mercaptoethanol and prepared for polyacrylamide gel electrophoresis. Samples were run on NuPAGE Bis–Tris gels (Fisher Scientific, Hampton, NH) and transferred to polyvinylidene difluoride (PVDF) membranes (Fisher Scientific, Hampton, NH) using the iBlot® 2 Dry Blotting System (Fisher Scientific, Hampton, NH). Washed PVDF membranes were blocked and then probed with the following primary antibodies: procaspase-1 + p10 + p12 [EPR16883] (Abcam, Cambridge, MA), β-actin (AC-74, Sigma-Aldrich, St. Louis, MO) and NLRP3 [Cryo-2] (AdipoGen Life Sciences, San Diego, CA). Bound primary antibodies were detected using HRP-labeled secondary antibodies and membranes were imaged with an Azure c300 imager (Azure Biosystems, Dublin, CA). Bands were quantitated using ImageJ software.

Ambrus-Aikelin G., Takeda K., Joetham A., Lazic M., Povero D., Santini A.M., Pranadinata R., Johnson C.D., McGeough M.D., Beasley F.C., Stansfield R., McBride C., Trzoss L., Hoffman H.M., Feldstein A.E., Stafford J.A., Veal J.M., Bain G, & Gelfand E.W. (2023). JT002, a small molecule inhibitor of the NLRP3 inflammasome for the treatment of autoinflammatory disorders. Scientific Reports, 13, 13524.

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