To analyze expression of recombinant fusion antigen in L. plantarum, cells from a 50 mL culture were harvested 3 h after induction (25 (link), 43 (link)) and resuspended in 500 μL PBS. Bacterial protein extracts were prepared by disruption in FastPrep tubes containing 1.5 g of glass beads (size ≤ 106 μm; Sigma-Aldrich), using a FastPrep® FP120 Cell Disrupter with a shaking speed of 6.5 m/s for 45 s. After 5 min incubation on ice the shaking process was repeated. The glass beads were removed by sedimentation and the protein extracts were transferred to a new tube. Proteins were separated by SDS-polyacrylamide gel electrophoresis using 10% Mini-Protean TGX Precast gels (BioRad) and transferred to a nitrocellulose membrane using the iBlotTM Dry Blotting System (Invitrogen). The proteins were detected using the SNAP i.d.® 2.0 Protein Detection System (Merck) using a specific monoclonal mouse anti-ESAT-6 antibody (Abcam) diluted 1:15000 and, subsequently, a polyclonal HRP-conjugated rabbit anti-mouse IgG (DAKO), diluted 1:7500. Proteins were visualized using the SuperSignal™ West Pico Chemiluminescent Substrate (Termo Fisher Scientific) and signals were documented using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems), following the manufacturer's instructions.
Azure c400 system
Manufactured by Azure Biosystems
Sourced in United States, Ireland
The Azure c400 system is a multi-channel imaging platform designed for high-throughput fluorescence and chemiluminescence detection. The system features a compact, modular design and supports a variety of microplate and gel imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Azurespot analysis software, by Azure Biosystems (8 mentions)
Ecl western blotting detection reagent, by GE Healthcare (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (3 mentions)
Coomassie brilliant blue r 250, by Avantor (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Glass bead, by Merck Group (1 mentions)
Snap i d 2.0 protein detection system, by Merck Group (1 mentions)
Hrp conjugated rabbit anti mouse igg, by Agilent Technologies (1 mentions)
Iblottm dry blotting system, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Un scan it software, by Silk Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti esat 6, by Abcam (1 mentions)
I blot transfer device, by Thermo Fisher Scientific (1 mentions)
M iggκ bp hrp, by Santa Cruz Biotechnology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
10 protocols using azure c400 system
1
Recombinant Fusion Antigen Expression in Lactobacillus
2
Investigating Cold Plasma-Induced Protein Expression
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HaCaT cells (3 × 105) were plated in 6-cm dishes and incubated with complete cell culture media which were pretreated with various conditions of He/Ar-CAPJ for 24 h. The treated cells were lysed with 100 μL RIPA reagent containing with protease and phosphatase inhibitors (Roch, Indianapolis, IN, United States). The cell lysates resolved by 10% SDS-PAGE and then performed the Western blot assay. The samples were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, United States) and immersed with 5% dehydrated skim milk to block non-specific protein binding. The membranes were incubated with primary antibodies as indicated in each experiment. The blots were then probed with the HRP-conjugated secondary antibody. The proteins of interest were detected using ECL Western blotting detection reagents (GE Healthcare, Piscataway, NJ, United States) and visualized by using Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, Dublin, CA, United States). The signal intensity of each protein was quantified with UN-SCAN-IT software (Silk Scientific Corporation, Orem, UT, United States) as described previously (Lin et al., 2019 (link)). These data were expressed as the mean ± standard deviation determined from three independent experiments.
3
Western Blot Analysis of ESAT-6
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The bacterial cells were harvested, lysed and loaded onto an SDS-PAGE gel as previously described [16 ]. The Bradford assay (Bio-Rad Laboratories, Hercules, CA) was used to determine the protein concentration of the cell free lysates. Approximately 0.8 µg crude protein extract was loaded onto the SDS-PAGE gel. After electrophoresis, proteins were blotted onto a nitrocellulose membrane using an iBlot™ Transfer Device (Invitrogen, Waltham, MA). Following the protein transfer, the SNAP i.d. 2.0 kit (Sigma-Aldrich) was used for antibody hybridization to the H56 antigen according to the manufacturer’s protocol. The mouse monoclonal antibody anti-ESAT-6 (ab26246, Abcam Inc, Cambridge, United Kingdom) primary antibody was diluted 1:2 000, and the secondary antibody m-IgGκ BP-HRP (Santa Cruz Biotechnology, Dallas, TX) was diluted 1:15 000. The proteins were visualized using the SuperSignal West Pico PLUS Chemiluminescent substrate (ThermoFisher Scientific) and the signals were imaged with an Azure c400 system (Azure biosystems, Dublin, CA).
4
Quantifying CDT Holotoxin Exposure in AGS Cells
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Each recombinant CdtA, CdtB, and CdtC was prepared and subjected to 12% SDS-PAGE, respectively. The gel was stained with Coomassie Brilliant Blue R-250 (Amresco, Solon, OH) for further analysis. AGS cells (5 × 105) were exposed to CDT holotoxin with various concentrations for different time durations. The cell lysates were prepared to resolve by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were probed with primary antibodies: RAGE and HMGB1 (Abcam, Cambridge, UK), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Millipore, Temecula, CA). The proteins of interests were detected using the ECL Western Blotting Detection Reagent (GE Healthcare, Piscataway, NJ) and visualized by using Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, Dublin, CA).
5
SLP Isolation and Western Blot Analysis
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Purified SLPs were boiled in SDS-PAGE sample buffer for 10 min and subjected to 10% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue R-250 (Amresco) for visualization of SLPs. In addition, the gel was transferred onto polyvinylidene difluoride membranes (PVDF, Millipore, Billerica, MA, USA). The membranes were blocked with TBST containing 5% skim milk for 1 h and then incubated with anti-SLPs antibody followed by incubation with HRP-conjugated secondary antibodies (Millipore) for 1 h. The proteins of interest were detected using ECL western blotting detection reagents (GE Healthcare, Chicago, IL, USA), and were visualized using Azure c400 system and AzureSpot Analysis Software (Azure Biosystems; Dublin, CA, USA) according to the manufacturer's instructions.
For monitoring caspase-1 and IL-1β maturation, total proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against IL-1β (R&D system, Minneapolis, MN, USA), precursor and p10 subunit of caspase-1 (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich, St. Louis, Missouri, USA). The expression of low-molecular-weight (LMW) surface layer proteins in the cell culture supernatant was also detected using rabbit anti-LMW SLP BAA 1805 serum (customized by Abnova, Taipei, Taiwan).
For monitoring caspase-1 and IL-1β maturation, total proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against IL-1β (R&D system, Minneapolis, MN, USA), precursor and p10 subunit of caspase-1 (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich, St. Louis, Missouri, USA). The expression of low-molecular-weight (LMW) surface layer proteins in the cell culture supernatant was also detected using rabbit anti-LMW SLP BAA 1805 serum (customized by Abnova, Taipei, Taiwan).
6
Western Blot Analysis of Lp_1261H2-DC in L. plantarum
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To analyze expression of Lp_1261H2-DC in L. plantarum, cells from a 50 mL culture were harvested 3 h after induction, cell-free protein extracts were prepared, and proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane, as described elsewhere [37 ]. The membrane was blocked by soaking in 5% (w/v) dry milk (Difco) in Tris Buffer Saline (TBS, 150 mM NaCl, 10 mM Tris-HCl; pH 7.4) supplemented with 0.1% (v/v) Tween-20 (Sigma-Aldrich) for 1 h at room temperature, followed by incubation with the anti-Hirep2 (anti-H2) primary antibody (produced in rabbit) in TBS with 3% (w/v) bovine serum albumin (BSA, Sigma-Aldrich), at 4°C, overnight. Subsequently, the blot was incubated with polyclonal HRP-conjugated goat anti-rabbit IgG (Invitrogen) diluted 1:5 000 in TBS containing 0.1% (v/v) Tween-20 and 5% (w/v) dry milk, for 1 h, at room temperature. Proteins were visualized using the SuperSignal™ West Pico Chemiluminescent Substrate (Termo Scientific) and signals were documented using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, Dublin, CA, USA), following the manufacturer’s instructions.
7
Investigating DNA Damage Response Pathways
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PC3-KD cells were seeded onto a 60-mm dish and incubated at 37 °C for 16 h, then treated with medium (mock), IR, HA-NPs accompanied by IR, CDT holotoxin accompanied by IR, and HA-CdtB-NPs accompanied by IR, respectively, for 24 and 48 h. Cell lysates were centrifuged at 12,000 rpm for 20 min at 4 °C. After quantification, samples were resolved by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). The polyvinylidene fluoride (PVDF) membrane was blocked and probed with primary antibodies against (ADP-Ribose) P polymerase (PARP), pH2AX, pATM, pCHK2, and β-actin in the condition of gentle shaking. After overnight incubation at 4 °C, the membrane was then probed with horseradish peroxidase-conjugated secondary antibody (Millipore) at room temperature for 1 h. The proteins of interest were detected using ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA) and were visualized using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, Dublin, CA, USA) by following the manufacturer’s instructions.
8
Graphene Effects on Duckweed Growth
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Lemna minor was obtained from the Department of Ichthyobiology, Fisheries and Aquaculture Biotechnology, Warsaw, Poland University of Life Sciences. Maintenance and cultivation of fronds were carried out at 20 °C in 6-well plates with Steinberg growth medium [26 (link)] under static conditions. Continuous white fluorescent lighting was used to provide a light intensity from the range of 8500–9500 lx. There were 10 plants per well. Graphene was introduced to the medium at increasing concentrations (5, 10, 20, 50 and 100 μg/mL). After 96 h, frond damage, surface area, biomass and root length were analyzed using a Leica DM750 microscope coupled with a Leica ICC50 digital camera and cellSens microscope imaging software (Olympus Corporation, Warsaw, Poland). The area of the fronds was analyzed by autofluorescence readings using the Azure C400 system (Azure Biosystems, Dublin, Ireland).
9
Western Blot Protein Detection Protocol
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Whole cell lysates were prepared, and total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The blots were incubated with 5% skim milk at room temperature for 1 h, then stained with primary antibodies, and then incubated with peroxidase-conjugated secondary antibody. The proteins of interest were detected by ECL Western blotting detection reagents (GE Healthcare, Piscataway, NJ, USA). The protein expression levels were analyzed using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, Dublin, CA, USA) following the manufacturer’s instructions [55 (link)].
10
Analyzing CdtB Localization and DNA Damage Markers
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PC3-KD cells were treated with 100 nM HA-NPs, CDT holotoxin, and HA-CdtB-NPs, followed by incubation for 0, 0.5, 1, 3, and 6 h. The nuclear proteins were isolated using a nuclear extraction kit (Pierce, Rockford, IL), as described previously [23] . CdtB level in the nuclear fraction was then analyzed by western blot assay.
Western blot analysis PC3-KD cells were seeded onto 60 mm dish and incubated in 37°C for 24 h, then treated with medium (mock), IR, HA-NPs accompanied by IR, CDT holotoxin accompanied by IR, and HA-CdtB-NPs accompanied by IR, respectively, for 24 h and 48 h. Cell lysates were centrifuged at 12,000 rpm for 20 min at 4°C. After quanti cation, samples were resolved by 12% SDS-PAGE and transferred onto polyvinylidene di uoride membranes (PVDF, Millipore, MA). The PVDF membrane was blocked and probed with primary antibodies against (ADP-Ribose) P polymerase (PARP), pH2AX, pATM, pCHK2, and β-actin in the condition of gentle shaking. After an overnight incubation at 4°C, the membrane was then probed with horseradish peroxidase-conjugated secondary antibody (Millipore) in room temperature for 1 h. The proteins of interests were detected using ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA) and were visualized using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, USA) by following the manufacturer's instructions.
Western blot analysis PC3-KD cells were seeded onto 60 mm dish and incubated in 37°C for 24 h, then treated with medium (mock), IR, HA-NPs accompanied by IR, CDT holotoxin accompanied by IR, and HA-CdtB-NPs accompanied by IR, respectively, for 24 h and 48 h. Cell lysates were centrifuged at 12,000 rpm for 20 min at 4°C. After quanti cation, samples were resolved by 12% SDS-PAGE and transferred onto polyvinylidene di uoride membranes (PVDF, Millipore, MA). The PVDF membrane was blocked and probed with primary antibodies against (ADP-Ribose) P polymerase (PARP), pH2AX, pATM, pCHK2, and β-actin in the condition of gentle shaking. After an overnight incubation at 4°C, the membrane was then probed with horseradish peroxidase-conjugated secondary antibody (Millipore) in room temperature for 1 h. The proteins of interests were detected using ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA) and were visualized using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, USA) by following the manufacturer's instructions.
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