For each experiment, proteins from the same number of cells per condition were extracted into SDS-sample buffer (0.5 M Tris; pH6.8; 12% SDS; 0.05% bromophenol blue). The lysates were tip sonicated and total proteins were loaded (100,000 cell equivalents), separated on 9% SDS–PAGE, followed by transfer to nitrocellulose membrane. Transfer efficiency was confirmed by Ponceau red staining, and membranes were blocked in blocking buffer (Tris-buffered saline, 0.1% Tween, and 2.5% nonfat dry milk) for 30 min before incubation with primary antibodies diluted in blocking buffer overnight at 4°C. After extensive washes in blocking buffer, membranes were incubated with appropriate secondary antibodies diluted in blocking buffer for 1 h at room temperature in the dark. After extensive washes in blocking buffer, membranes were scanned on an Odyssey 9120 Imaging System (LI-COR Biosciences), and protein bands were quantified using Image Studio Lite (LI-COR Biosciences).
Odyssey 9120 imaging system
Manufactured by LI COR
Sourced in United States
The Odyssey 9120 imaging system is a high-performance instrument designed for fluorescence-based detection and quantification of proteins and nucleic acids. It features a dual-channel detection system that allows for simultaneous imaging of two different fluorescent signals, enabling efficient and accurate analysis of complex biological samples.
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Lab products found in correlation
Irdye 800cw goat anti rabbit igg, by LI COR (3 mentions)
Image studio lite, by LI COR (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Sds page gel electrophoresis, by Bio-Rad (2 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Protein extraction kit, by Beyotime (2 mentions)
Mini trans blot module, by Bio-Rad (1 mentions)
Powerpac basic power supply, by Bio-Rad (1 mentions)
Mini protean tetra cell, by Bio-Rad (1 mentions)
Complete mini proteasome inhibitors, by Merck Group (1 mentions)
Phosstop, by Merck Group (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Tris glycine sds running buffer, by Thermo Fisher Scientific (1 mentions)
Tris glycine transfer buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Anti v5, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
14 protocols using odyssey 9120 imaging system
1
Western Blotting Quantification Protocol
2
Western Blot Protein Analysis Protocol
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Samples were mixed with 4X Laemmli buffer and boiled at 95°C for 5 min. 10% polyacrylamide gels were hand-casted and loaded with 20 μg of sample. The gels were run using a Mini-PROTEAN Tetra Cell (BioRad #1658001) and PowerPac Basic Power Supply (BioRad #1645050) at 100V for approximately 90 min. Gels were prepared for transfer to PVDF membranes, and the transfer was run using a Mini Trans-Blot Module (BioRad #1703935) at 400 mV for 60 min. The membranes were removed and blocked in 5% milk dissolved in TBST for 1 h. They were then incubated overnight with primary antibody [anti-tau (1:500,000), anti-GAPDH (1:1,000), and anti-Histone H3 (1:1,000)] overnight at 4°C on a shaker. The membranes were washed and incubated with secondary antibody [anti-rabbit AlexaFluor600 (1:5,000) and anti-mouse AlexaFluor800 (1:10,000)] for 1 h at room temperature. Imaging was done using a Licor Odyssey 9120 Imaging System. Quantification was done using ImageJ.
3
Protein Quantification and Immunoblotting
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Cell and exosome samples were lysed in RIPA buffer (Thermo Fisher Scientific, catalog no. 89900) supplemented with PhosSTOP (Sigma-Aldrich, catalog no. 4906837001) and Complete Mini proteasome inhibitors (Sigma-Aldrich, catalog no. 05892791001). Total protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, catalog no. 23225). A total of 30 μg total protein was loaded for PC3, TRAMP-C2, and MC38 cells and exosomes, and 40 μg for CT26 and B16F10 cells and exosomes. The cell and exosome protein samples were subjected to immunoblotting by following the manufacturer recommended protocols for the antibodies used, followed by imaging using the LI-COR Odyssey 9120 imaging system.
4
Western Blot Protein Analysis Protocol
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2 d after transfection, cells were lysed with 200 µl/well of 1× Laemmli buffer, and lysates were gently denatured at 65°C for 30 min in Eppendorf tubes before SDS-PAGE. 40 µl of each sample was loaded onto 4–12% Tris-glycine mini-gel (Thermo Fisher Scientific). After SDS-PAGE at 110 V in Tris-glycine SDS running buffer (Thermo Fisher Scientific), proteins were transferred to nitrocellulose membranes (0.45 µm pore size; Thermo Fisher Scientific) at 100 V for 1 h in Tris-glycine transfer buffer (Thermo Fisher Scientific) containing 5% methanol (vol/vol). Nonspecific antibody binding was blocked with Odyssey Blocking Buffer (LI-COR) for 1 h at room temperature. Primary antibodies were diluted in 5% BSA-PBS solution (wt/vol) and incubated at 4°C overnight. Primary antibodies were diluted in the following ratio: α-tubulin 1:2,000, α-OSBPL5 1:500, and α-ORP8 1:500. After three washes (for 10 min each) with 0.1% Tween 20–containing PBS solution (vol/vol), fluorescence-conjugated secondary antibodies (diluted 1:5,000) were incubated in PBS-containing 5% BSA at room temperature for 1 h. Fluorescence signal was detected with the Odyssey 9120 imaging system (LI-COR), and the images obtained from individual wavelengths were presented with grayscale by using OdysseyV3.0 software.
5
Western Blot Analysis of Protein Expression
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For each experiment, proteins from the same number of cells per condition were extracted into SDS-sample buffer (0.5 M Tris at pH 6.8, 12% SDS, 0.05% bromophenol blue). The lysates were tip-sonicated and total proteins loaded (100,000 cell equivalents), separated on 9% SDS-PAGE, and transferred to nitrocellulose membrane. Transfer efficiency was confirmed by Ponceau red staining, and membranes were blocked in blocking buffer (Tris-buffered saline, 0.1% Tween, 2.5% nonfat dry milk) for 30 min before incubation with primary antibodies diluted in blocking buffer overnight at 4°C. After three washes in blocking buffer, membranes were incubated with appropriate secondary antibodies diluted in blocking buffer for 1 h at room temperature in the dark. After three washes in blocking buffer, membranes were scanned on an Odyssey 9120 imaging system (LI-COR Biosciences), and protein bands were quantified using Image Studio Lite (LI-COR Biosciences) and analyzed with Microsoft Excel and GraphPad Prism software.
6
Crystal Violet Cell Staining Protocol
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Cells were seeded in 96-well transparent plates and manipulated as indicated in the figures. At the end-point, cells were washed twice with PBS and fixed with ice-cold methanol for 10 min at −20 °C. After removing the fixative, plates were equilibrated to room temperature and fixed cells were incubated in crystal violet staining solution (0.5 g crystal violet powder, 25 ml methanol and 75 ml distilled water) for 10 min at room temperature. Plates were then washed with distilled water, air-dried overnight and scanned using Odyssey 9120 imaging system (LI-COR). Plates corresponding to consecutive time points within a complete experiment were scanned at the same time. Composite images were generated using ImageJ-win64 software, with splicing indicated by a grey dotted line.
7
Western Blot Protein Detection Protocol
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Western blot samples were separated on 4–22% gradient SDS-PAGE gels, transferred to PVDF membrane (30 V, overnight) blocked with Licor blocking buffer in TBST, and probed with anti-His (Takara, 1:1,000), or anti-V5 (Invitrogen, 1:500) primary antibodies rocking for 16 h at 4°C. After rinsing three times with TBST, the membranes were than incubated with Li-Cor IRDye 800CW Goat anti-rabbit or anti-mouse IgG (1:10,000) secondary antibody, rocking for 1 h at room temperature, and washed three times for 5 min in TBST. The membrane was then scanned using a Li-Cor Odyssey 9,120 Imaging System. If antibody overlays were desired, after imaging, the additional antibody was added to the membrane and incubated for 1 h at room temperature. After incubation, an appropriate secondary antibody was added, and the blot visualized as described above.
8
Analyzing PI-103 Drug Effects in Irradiated U87 Cells
9
Fluorescence-based Western Blotting Protocol
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Western blotting was performed essentially as described previously6 (link). Briefly, Immobilon-FL PVDF membranes (Merck Millipore) were pre-activated with MeOH and used for wet blotting for 1 h at 100 V. Blocking was accomplished with casein blocking buffer (Sigma) for 30 min at room temperature while shaking on a roller shaker. Then, the primary antibody solution in PBS with 0.1% (v/v) Tween-20 and 1 × casein blocking buffer (antibody incubation buffer) was applied on the blot and incubated for 1 h at room temperature while shaking on the roller shaker. The primary antibodies used were streptavidin-IRDye 680LT (LI-COR Biosciences), polyclonal rabbit anti-HA (H6908, Sigma-Aldrich), mouse anti-actin (ab8224, Abcam) and a home-made rabbit anti-DARPin serum. Secondary antibodies were goat anti-rabbit Alexa Fluor 680 (Invitrogen), donkey anti-mouse IRDye800 CW (LI-COR Biosciences) and goat anti-rabbit IRDye800 CW (LI-COR Biosciences). When incubating with the fluorescently labelled streptavidin (IRDye 680LT), also 0.1% (w/v) SDS was included to reduce non-specific binding of streptavidin. Blots were either incubated with streptavidin-IRDye 680LT and anti-DARPin serum together, or with the anti-HA and anti-actin antibodies together. Detection was accomplished using an Odyssey 9120 imaging system (LI-COR Biosciences).
10
Evaluating Protein Markers in VaD Mouse Hippocampus
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To determine the effect of EA in the hippocampus of VaD mice at protein level, we divided a further 18 rats (n = 6 each group) into 3 groups: Sham, BCCAo, and EA.
RIPA lysis buffer containing a mixture of complete protease inhibitors (Roche Diagnostics) was used for sample homogenizing. BCA method was used to determine protein concentration. The primary antibodies include anti-JNK-3 (Santa Cruz, sc-507), anti-AP-1 (Proteintech, 50599-2-Ig), anti-P53 (Proteintech, 12789-1-AP), anti-Bcl-2 (Proteintech, 12789-1-AP), anti-Bax (Proteintech, 50599-2-Ig), anti-Caspase-3 (Proteintech, 19677-1-AP-150),and GAPDH (sigma, P7769-5MG). Secondary antibodies were used to incubated membranes for 2 hours at room temperature. Membranes were scanned and analyed by Odyssey 9120 imaging system (LICOR, Inc).
RIPA lysis buffer containing a mixture of complete protease inhibitors (Roche Diagnostics) was used for sample homogenizing. BCA method was used to determine protein concentration. The primary antibodies include anti-JNK-3 (Santa Cruz, sc-507), anti-AP-1 (Proteintech, 50599-2-Ig), anti-P53 (Proteintech, 12789-1-AP), anti-Bcl-2 (Proteintech, 12789-1-AP), anti-Bax (Proteintech, 50599-2-Ig), anti-Caspase-3 (Proteintech, 19677-1-AP-150),and GAPDH (sigma, P7769-5MG). Secondary antibodies were used to incubated membranes for 2 hours at room temperature. Membranes were scanned and analyed by Odyssey 9120 imaging system (LICOR, Inc).
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