An equal quantity of protein in 16 μL was loaded per well of pre-cast mini-protean TGX gels 4–20% gels (Biorad), with one gel per timeseries. Electrophoresis was applied according to the manufacturers protocol with a tris–glycine-SDS running buffer (Biorad). Protein transfer onto nitrocellulose membranes (Biorad) was performed using the trans-blot turbo mini kit (Biorad). The membranes were allowed to dry at room temp for 1 h prior to quantifying the total protein using the Li-Cor total protein stain protocol and Odyssey blot-imager (Li-Cor). The membranes were blocked using 5% non-fat milk powder (Marvel) in Tris-buffered saline (TBS) for 1 h at room temperature before incubating overnight at 4 °C with primary antibody (rabbit anti-bmal-1, abcam ab93806) diluted 1:3000 in 2.5% milk in TBS + 0.1% tween-20. After washing, membranes were incubated for 1 h at room temp with secondary antibody, donkey anti-rabbit IRDye 800CW (Licor, 925–32,213) diluted 1:10,000 in TBS + 0.2% tween-20. The membranes were washed extensively before imaging with the Li-Cor Odyssey blot-imager. The 800 nm fluorescence signal from BMAL-1 was quantified using the Image Studio Lite software (Li-Cor Bioscience) and normalised to total protein. Representative Western blot images are presented in Fig. S1 in the supplementary material.
Odyssey blot imager
Manufactured by LI COR
Sourced in United States
The Odyssey Blot Imager is a laboratory instrument designed for the detection and quantification of proteins and other biomolecules on Western blots. It utilizes near-infrared fluorescence technology to provide sensitive and accurate results.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (4 mentions)
Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (4 mentions)
Immobilon fl pvdf membranes, by LI COR (4 mentions)
Image studio lite software, by LI COR (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Mini protean tgx 4 20 sds page gel, by Bio-Rad (2 mentions)
Nitroceullose membranes, by Bio-Rad (2 mentions)
Trans blot turbo, by LI COR (2 mentions)
Intercept buffer, by LI COR (2 mentions)
Iblot2 system, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Iblot2, by Thermo Fisher Scientific (2 mentions)
Af 201 na, by Takara Bio (2 mentions)
Tub 2, by Thermo Fisher Scientific (2 mentions)
Alexfluor 680 conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (2 mentions)
A2091, by ABclonal (2 mentions)
A9961, by ABclonal (2 mentions)
A5561, by ABclonal (2 mentions)
A28183, by Thermo Fisher Scientific (2 mentions)
26 protocols using odyssey blot imager
1
Western Blot Analysis of BMAL-1 Protein
2
Caspase-11 Expression in Macrophages
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Lysates were prepared from Casp11+/– and Casp11–/– mouse bone marrow-derived macrophages and clarified by spinning at 16,100×g for 10 min at 4°C. Clarified lysates were denatured in SDS loading buffer. Samples were separated on NuPAGE Bis-Tris 4–12% gradient gels (Thermo Fisher) following the manufacturer’s protocol. Proteins were transferred onto Immobilon-FL PVDF membranes at 375 mA for 90 min and blocked with Odyssey blocking buffer (Li-Cor). Proteins were detected on a Li-Cor Odyssey Blot Imager using an anti-Caspase-11 primary antibody (cone 17D9) and Alex Fluor-680 conjugated secondary antibody (Invitrogen).
3
Western Blot Analysis of Protein Lysates
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Total cell lysates were prepared in RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40; 0.1% SDS) or cell lysis buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1% Triton-X-100) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma) and protein concentrations were determined by BCA assay kit (Thermo Scientific, Waltham, MA). Thirty micrograms of total cell lysates were subjected to 4–20% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked in 20 mM Tris-HCl, 137 mM NaCl, and 0.2% Tween 20 (pH 7.6) containing 5% nonfat milk. The membrane was immunoblotted with primary antibodies at 4 °C overnight followed by secondary antibody conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA) for 1 hr. The membrane was exposed on X-ray film using ECL Western blot detection reagents (Pierce Biotechnology, Rockford, IL) or digitally captured using Odyssey blot imager (LI-COR, Lincoln, NE). Raw uncropped images with molecular size marker were displayed in the supplementary section (Figs S4 –S9 ). Immunoblot band density was measured by ImageJ program (National Institute of Health) and normalized by the intensity of loading control (Tubulin or Actin) as indicated.
4
Western Blotting Protocol for Protein Analysis
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Equal amounts of lysates were loaded onto Mini-Protean TGX (4-20%) SDS-PAGE gels (BioRad). All blots except phospho-MTFR1L were transferred to nitroceullose membranes (BioRad) with a Trans-Blot Turbo and blocked for one hour in Intercept Buffer (Li-COR). Membranes were then incubated in their respective primary antibodies in blocking buffer at 4°C overnight. Blots probing for phospho-MTFR1L we transferred to polyvinylidene difluoride membranes (PVDF, Immobilion) and blocked in 5% fat-free dry milk in TBS-T for 1 hour before primary antibody incubation for four days at 4°C. Membranes were incubated in Li-Cor fluorescence-coupled secondary antibodies for 1 hour at room temperature prior to visualization with a Li-Cor Odyssey Blot Imager.
5
Immunoprecipitation and Western Blot Analysis
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Cells (4 × 106) were plated on 100 mm tissue-grade culture plates and primed overnight with 1 μg/ml IgE. Cells were stimulated with respective antigen or mock for the indicated times at 37°C in Hank’s buffered saline. Cells were rinsed in cold phosphate-buffered saline and lysed on ice for 20 min with NP-40 lysis buffer (150 nM NaCl, 50 mM Tris, 1% NP-40) in the presence of Halt Protease and Phosphatase inhibitors (Thermo Fisher Scientific). Lysates were cleared by centrifugation at 13,000 × g for 10 min at 4°C. Protein concentrations were determined by BCA assay (Thermo Fisher Scientific), and 25 μg was loaded into polyacrylamide gels. Whole lysates or immunoprecipitated samples were boiled with reducing sample buffer, except for lysates used for the detection of FcεRIγ phosphorylation where nonreducing sample buffer was used to preserve the γ-dimer. Proteins were transferred from the SDS–PAGE gel to nitrocellulose membranes using the iBlot2 system (Life Technologies). Membranes were blocked for 30 min in 3% bovine serum albumin (BSA)/0.1% Tween-20/ tris buffered saline (TBS) and probed overnight with primary antibodies at 4°C. HRP-conjugated secondary antibodies were used for detection and incubated with membranes for 1 h at RT. Membranes were imaged on the Odyssey Blot Imager (Li-Cor). Analysis was performed using ImageStudio software.
6
Protein Extraction from 2D and 3D Cell Cultures
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Cells were rinsed twice with HBSM and lysed by scraping into Laemmli buffer. For analysis of cells growing in 3D conditions, cells growing on 0.8 mg/ml collagen I were rinsed twice with HBSM and then incubated two minutes at room temperature (RT) with Laemmli buffer. Lysate was collected from the surface of the 3D matrix by pipetting. Protein concentrations of lysates were normalized using the RED 660 Protein Assay (G Biosciences) prior to SDS-PAGE and immunoblotting. Blots were analyzed using a LiCOR Odyssey blot imager.
7
Quantitative Protein Determination via SDS-PAGE
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Absorption spectroscopy indicated, via a 260-nm/280-nm ratio, that despite the two steps of affinity purification under high salt concentration and size exclusion chromatography, nucleic acid copurifies with Rep; thus, protein concentration was determined using quantitative SDS-PAGE analysis. The PAGE was quantitated using the Li-Cor Odyssey blot imager equipped with Image Studio software version 5.0. The concentration of Rep (35.8 kDa) was determined by comparing its band intensity to that of carbonic anhydrase (29 kDa; Millipore Sigma) with known concentration.
8
Quantitative Protein Analysis of Brain Samples
9
Western Blot Analysis of Protein Expression
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Liquid-nitrogen-frozen ears were ground and re-suspended in Laemmli buffer. Whole cell extracts were prepared in CelLytic MT Cell Lysis Reagent (Millipore Sigma) and diluted in Laemmli buffer. Protein concentrations were determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. A total of 5% of dithiothreitol was added to the extracts and then heated at 95 °C for 5 min. Reduced extracts were resolved by SDS-PAGE and transferred onto nitrocellulose membrane for immunoblot analysis. Transferred membranes were blocked with 5% non-fat milk in TBS supplemented with 0.05% Tween-20 and then probed with primary antibodies (1:1000 dilution) except COL17 NC16a antibody (1:2000 dilution) in blocking buffer at 4 °C overnight. Membranes were then incubated with 1:3000 dilution of secondary antibodies in blocking buffer for 1 h. Immunoblots were visualized using an Odyssey Blot Imager (LI-COR Biosciences, Lincoln, NE, USA) and quantified using ImageJ. Uncropped and unprocessed scans of the blots are included in the Source Data file.
10
Western Blot Analysis of SP110 and SP140
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Samples were lysed in RIPA buffer with protease inhibitor cocktail (Roche) to obtain total protein lysate and were clarified by spinning at ~16,000×g for 30 min at 4°C. Clarified lysates were analyzed with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's specification and diluted to the same concentration and denatured with SDS-loading buffer. Samples were separated on NuPAGE Bis–Tris 4–12% gradient gels (Thermo Fisher Scientific) following the manufacturer’s protocol. Gels were transferred onto ImmobilonFL PVDF membranes at 35 V for 90 min and blocked with Odyssey blocking buffer (Li-Cor). Proteins were detected on a Li-Cor Odyssey Blot Imager using the following primary and secondary antibodies. Rabbit anti-SP110 or SP140 serums were produced by Covance and used at 1:1000 dilution. Hybridoma cells expressing monoclonal anti-SP110 antibody were from the lab of I. Kramnik. Antibodies were produced in-house as previously described (Ji et al., 2019 (link)) and used at 100 ng/mL. Alexa Fluor 680-conjugated secondary antibodies (Invitrogen) were used at 0.4 mg/mL.
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