Briefly, for western blot analysis, 50 µg of protein was loaded onto a 12% polyacrylamide gel Bolt TM 8% Bis- Tris Plus Invitrogen (Thermo Fisher Scientific, CA, USA) followed by electrotransfer to nitrocellulose membrane iBlot® Gel Transfer Stacks Nitrocellulose Regular (Thermo Fisher Scientific, Kiryat Shmona, Israel) using iBLOT Invitrogen transfer (Life Technologies, Israel). Subsequently, the membrane was blocked in Odyssey Blocking Buffer (Licor, Milan, Italy) for 1 h at room temperature. After blocking, the membrane was washed three times in PBS for 5 min and incubated with primary antibodies against HO-1 (1:1000) (anti-rabbit, Cat. No. BML-HC3001-0025, Enzo Life Sciences, Milan, Italy) and β-actin (1:1000) (anti-mouse, Cat. No. 69879, Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. The following day, membranes were washed three times in PBS for 5 min and incubated with Infrared anti-mouse IRDye800CW (1:5000) and anti-rabbit IRDye700CW secondary antibodies (1:5000) in PBS/0.5% Tween-20 for 1 h at room temperature. All antibodies were diluted in Odyssey Blocking Buffer. The blots were visualized using an Odyssey Infrared Imaging Scanner (Licor, Milan, Italy) and protein levels were quantified by densitometric analysis of antibody responses. Data were normalized to the total protein levels of β-actin.
Odyssey infrared imaging scanner
Manufactured by LI COR
Sourced in Italy, United States, Canada
The Odyssey Infrared Imaging Scanner is a laboratory equipment product designed for protein and nucleic acid detection and quantification. It utilizes infrared fluorescence technology to capture and analyze images.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (11 mentions)
Trans blot turbo, by Bio-Rad (10 mentions)
Miniprotean tgxtm, by Bio-Rad (10 mentions)
Transblot se semi dry transfer cell, by Bio-Rad (10 mentions)
Ripa lysis buffer, by Beyotime (7 mentions)
Bml hc3001 0025, by Enzo Life Sciences (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Image studio software, by LI COR (2 mentions)
Black 96 well plate, by Greiner (2 mentions)
Ab23707, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Mouse anti gapdh monoclonal antibody, by Merck Group (2 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (2 mentions)
Bca kit, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Odyssey clx, by LI COR (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
36 protocols using odyssey infrared imaging scanner
1
Western Blot Quantification of HO-1 Protein
2
Reverse Transfection and Western Blot Analysis
3
Quantitative Immunoblotting Analysis of Signaling Pathways
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Cells were starved overnight before treatment. Whole cell lysates were obtained using RIPA buffer (Sigma) supplemented with phosphatase and protease inhibitors (Roche, Germany). Protein expression was analysed by western blotting as described previously19 (link). Briefly, antibodies used were as follows: phospho-CREB, CREB, phospho-ERK1/2, ERK1/2, phospho-p38, p38, and phospho-PKA substrates (Cell Signaling, MA, USA), and β-Tubulin (Santa Cruz Biotechnology). Fluorophore conjugate antibodies were obtained from Invitrogen. Signals were visualized and recorded with an Odyssey Infrared Imaging scanner (LI-COR Biosciences, NE, USA). Immunoblot densitometry analysis on every band was calculated using Image Studio Software (LI-COR Biosciences). Phosphorylation and total protein densitometry values were normalized to β-Tubulin signal.
4
Quantitative Analysis of DNA Damage Response
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ICW analysis was performed as previously described.61 (link) Cells were seeded at 2 × 104 cells/well and treated with increasing doses of CDT (5, 50, 500 ng/ml) or CDTmut (500 ng/ml) in triplicates in 96-well plates. After 24 h, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at RT. Upon washing with PBS, paraformaldehyde was neutralized using NH4Cl (20 mM). Next, cells were permeabilized with 0.2% Triton X-100 in PBS. Following washing with PST (PBS, 0.2% Triton X-100, 2% FCS), cells were blocked with MAXBlock Blocking Medium (Active Motif) supplemented with RNase A (Sigma) and cOmplete Protease Inhibitor (Roche) for 1 h at RT. Cells were then incubated with γH2AX antibody (1 : 200 in PST) for 2 h at RT. As secondary antibody, an infrared fluorescent dye-conjugated antibody (1 : 1000 in PST; 800 nm; LiCor Biosciences, Bad Homburg, Germany) was used for 1 h at RT. For DNA labeling, RedDot2 (1 : 1000 in PST; Biotum, Fremont, CA, USA) was added to the secondary antibody solution. Measurements were performed using an Odyssey Infrared Imaging Scanner (Li-Cor Biosciences). DNA and γ-H2AX staining were simultaneously visualized. Statistical analysis in the Image Studio Light (Version 5.2; Li-Cor Biosciences) was performed to determine the x-fold increase in γ-H2AX signal normalized to the DNA content per well in relation to the control.
5
HEK-293T Cell Transfection and Protein Analysis
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Cells were transfected in 6-well plates using Lipofectamine 2000 reagent, again using the 1-day protocol described above, with 1 μg of each indicated plasmid. The transfecting DNA complexes in each well were incubated with 8 × 105 HEK-293T cells suspended in 3000 μl DMEM + 10% FBS and incubated overnight at 37°C in 5% CO2. Transfected cells were lysed in 100 μl 1 × PLB. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Protran), which were incubated at 4°C overnight with primary antibodies. Immunoreactive bands were detected on membranes after incubation with appropriate fluorescently labelled secondary antibody using a LI-COR Odyssey® Infrared Imaging Scanner.
6
Dual Luciferase Assay in HEK293T Cells
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HEK293T cells were transfected in six-well plates using Lipofectamine 2000 reagent, again using the 1-d protocol described above, with 1 µg of each indicated plasmid. The transfecting DNA complexes in each well were incubated with 1 × 106 cells suspended in 3000 µL DMEM plus 10% FBS and incubated overnight at 37°C in 5% CO2. Transfected cells were lysed in 100 µL 1× PLB and 10 µL each lysate assayed by dual luciferase assay. Proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes (Protran), which were incubated at 4°C overnight with goat anti-Firefly (Promega) and mouse anti-β-actin (Sigma). Immunoreactive bands were detected on membranes after incubation with appropriate fluorescently labeled secondary antibody using a LI-COR Odyssey Infrared Imaging Scanner. ImageStudio software was used for densitometry. Firefly intensities were calculated relative to β-actin intensities and readthrough efficiencies determined as a percent of the corresponding normalized in-frame controls. Mean and standard deviations of relative protein intensities were determined from three biological replicates.
7
Western Blot Analysis of DDAH-1, SREBP1c
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Tissue homogenates (30 μg proteins) containing a protease-inhibitor cocktail were loaded onto 12% SDS-polyacrylamide (SDS-PAGE) gels and subjected to electrophoresis (120 V, 90 min). The separated proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After transfer, the blots were incubated with Li-COR blocking buffer for 1 h, followed by overnight incubation with a 1 : 1.000 dilution of primary antibodies directed against DDAH-1 [Calbiochem EMD Biosciences (Darmstadt, Germany)], SREPB1c (SantaCruz Biotechnology, Santa Cruz, CA, USA) and β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA). After washing with TBS, the blots were incubated for 1 h with the secondary antibody (1 : 1.000). Protein detection was carried out using a secondary infrared fluorescent dye-conjugated antibody absorbing at λ 800 and λ 700 nm. The blots were visualized using an Odyssey Infrared imaging scanner (LI-COR Biosciences) and quantified by densitometric analysis performed after normalization with β-actin. Results are expressed as arbitrary units (A.U.).
8
Immunoblotting of Cell Signaling Proteins
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Tissues and cells of human were lysed in radioimmunoprecipitation assay buffer (Beyotime) containing a protease and phosphatase inhibitor cocktail (K1015, APExBIO). The protein concentration was assayed by the BCA method. The extracted or enriched protein samples were separated by denaturing 10% SDS-polyacrylamide gel electrophoresis and transferred into polyvinylidene fluoride membranes. After being blocked by 5% bovine serum albumin (BSA) for 120 min at room temperature, the membranes were incubated with primary antibodies from abcam and Cell Signaling Technology against GAPDH (ab8245, 1:8,000), mTOR (ab2732, 1:1,500), AKT (ab18785, 1:1,000), p-mTOR (ab109268, 1:1,500), p-AKT (ab38449, 1:1,000), P70S6K (#2708, 1:1,000), p-P70S6K (#9205, 1:1,000), and CAMK1D (ab172618, 1:1,500) overnight at 4°C. After washing three times, the blots were further incubated with goat anti-rabbit IRDye 800CW preadsorbed secondary antibody (1:10,000; Abcam; ab216773). The images were detected with an Odyssey infrared imaging scanner (LI-COR, USA).
9
Western Blot Analysis of Immune Signaling
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Briefly, for western blot analysis 50 μg of proteins were loaded onto a 12% polyacrylamide gel Mini- PROTEAN® TGXTM (BIO-RAD, Milan, Italy) followed by electrotransfer to nitrocellulose membrane Trans- Blot® TurboTM (BIO-RAD) using Trans- Blot® SE Semi-Dry Transfer Cell (BIO-RAD). Subsequently, membrane was blocked in Odyssey Blocking Buffer (Licor, Milan, Italy) for 1 h at room temperature. After blocking, membrane was washed three times in PBS for 5 min and incubated with primary antibodies against human MyD88, TLR4 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), overnight at 4 °C. Next day, membranes were washed three times in PBS for 5 min and incubated with Infrared anti-mouse IRDye800CW (1:5000) and anti-rabbit IRDye700CW secondary antibodies (1:5000) in PBS/0.5% Tween-20 for 1 h at room temperature. All antibodies were diluted in Odyssey Blocking Buffer. The blots were visualized using Odyssey Infrared Imaging Scanner (Licor, Milan, Italy) and protein levels were quantified by densitometric analysis of antibody responses. Data were normalized to protein levels of β-actin.
10
Quantification of DNA Double-Strand Breaks
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Quantification of DNA DSBs was performed using the In Cell Western assay as described [25 ]. Caco-2 cells were dispensed (1 × 105 cells/well) in a black 96 well plate (Greiner Bio-One) and incubated at 37 °C in 5% CO2 atmosphere. After 24 h, Caco-2 cells were infected at MOI 50 with E. coli strains. After 4 h of infection, the cells were washed three times with PBS and incubated for 3 h in cell culture medium supplemented with 200 μg/ml gentamicin. Cells were fixed (4% paraformaldehyde), permeabilized, blocked and then incubated overnight with rabbit monoclonal anti-γ-H2AX (BioLabs) at 1/200 dilution. Secondary antibody IRDye™800CW goat anti-rabbit (Biotium, Wisconsin, United States) was applied simultaneously with 1/500 dilution of RedDot™2 (Biotium) for DNA labeling. The DNA and γ-H2AX were visualized using Odyssey® infrared imaging scanner (LI-COR model 9120, Québec, Canada) with red denoting RedDot™2 and green for IRDye™800CW goat anti-rabbit. Images were processed using Image Studio Ver3.1 software.
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