Whole cell extracts were prepared by incubating cells with RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% NaDeoxycholate, 0.1% SDS, 25 mM DTT, 10 mM EDTA, 1 mM PMSF) plus 1 μl each of protease and phosphatase inhibitor mixes (Sigma, St Louis, MO) per 100 μl buffer. After removing cell debris by centrifugation, protein concentration was determined using a micro BCA assay (Pierce/ThermoScientific, Rockford, IL). Extracts (20–50 μg per well) were separated on a Criterion TGX gel (Biorad, Hercules, CA), transferred to PVDF membrane and probed with rabbit anti-FLI1 (Santa Cruz Biotechnology, Dallas, TX) or rabbit anti-βactin (Cell Signaling Technology, Danvers, MA) antibodies. Anti-FLI1 was detected with an anti-rabbit biotin antibody (Pierce/ThermoScientific) followed by an AlexaFluor-647 streptavidin-conjugated antibody (Life Technologies, Grand Island, NY). Blots were stripped and reprobed with anti-βactin and detected with a goat anti-rabbit AlexaFluor-647 antibody (Life Technologies). Blots were scanned on an Odyssey Infrared Imaging system and software (LI-COR, Lincoln, NE).
Odyssey infrared imaging system and software
Manufactured by LI COR
Sourced in United States, Germany
The Odyssey infrared imaging system and software is a versatile tool for detecting and quantifying proteins, nucleic acids, and small molecules in a variety of applications. The system utilizes infrared fluorescence technology to provide high-sensitivity and high-resolution imaging of protein and nucleic acid samples. The accompanying software allows for comprehensive analysis and data management.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Irdye 800, by LI COR (3 mentions)
Irdye 680, by LI COR (2 mentions)
Odyssey 680 800 nm secondary conjugates, by LI COR (2 mentions)
Ecl western blotting detection system, by GE Healthcare (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Alexa fluor 700 conjugated secondary antibody goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Irdye800 conjugated goat anti rabbit igg, by Rockland Immunochemicals (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Rabbit anti fli1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Criterion tgx gel, by Bio-Rad (1 mentions)
Micro bca assay, by Thermo Fisher Scientific (1 mentions)
Anti adiponectin, by Abcam (1 mentions)
Anti c myc 9e10, by Merck Group (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Pvdf membrane, by LI COR (1 mentions)
Odyssey buffer, by LI COR (1 mentions)
Tissue lysis buffer, by Beyotime (1 mentions)
29 protocols using odyssey infrared imaging system and software
1
Western Blotting of FLI1 and β-actin
2
Analyzing Adiponectin Multimer Separation
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Media were removed from cells and centrifuged for 5 min at 5000 g , 4°C to pellet cellular debris. The supernatant was used for sucrose gradient separation of adiponectin multimers or ammonium sulphate precipitation for non-denatured Western blot analysis as previously described [4 (link),16 (link)]. For sucrose gradients, 50 µl of unconcentrated media were loaded onto the sucrose gradient. Whole cell lysates were boiled in SDS/PAGE loading dye with DTT for 20 min, then separated by SDS/PAGE and subsequently transferred to Immobilon-FL membrane, 0.45 μm (Merck-Millipore, Bayswater, VIC, Australia) for Western blot analysis. Adiponectin monomers and trimers were prepared as previously characterized [17 (link)]. Monomers were reduced with DTT and boiled whereas trimers were only reduced. Sample loading was calculated by loading the volume of media relative to equal volumes of RNA; hence, normalization was calculated based on RNA. Primary antibodies included anti-carboxy terminus GLT25D1 (C-16) (Santa Cruz), anti-PLOD3 purified MaxPab (Abcam), anti-adiponectin (Abcam), in-house anti-adiponectin [18 (link)], anti-β-tubulin (Sigma–Aldrich) and anti-c-Myc (9E10) (Sigma–Aldrich). Analysis and quantitation of Western blots were conducted using the Odyssey Infrared Imaging System and Software (LI-COR Biotechnology, Lincoln, NE, U.S.A.).
3
Western Blot Analysis of Protein Expression
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For the western blot analysis, the cells were lysed in RIPA buffer containing 0.3 M NaCl, 1% sodium desoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 20 mM Tris-HCL (pH 8.0), 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein (25 μg) was fractionated by 15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked in Odyssey buffer (Li-COR Biosciences, Lincoln, NE, USA) for 1 h. The protein expression levels were determined by immunoblotting with the polyclonal antibodies anti-hLifeguard β-isoform (1:200 dilution; generated in our laboratory) and monoclonal anti-hNGF-R, TrK-A/B, Act-p, actin (all used at 1:500; Abcam, Cambridge, UK) at 4°C overnight. To quantify the protein expression levels, Odyssey 680/800 nm secondary conjugates were used, and the PVDF membranes were analyzed using the Odyssey infrared imaging system and software (Li-COR Biosciences).
4
Western Blot Protein Analysis
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Western blot analysis was performed as described previously [58 ]. Proteins from isolated Sertoli cells and purified spermatocytes were electrophoresed under reducing conditions in 12% SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were blocked in 5% BSA, incubated overnight at 4°C with the primary antibody, and incubated with the IRDye 680 or IRDye 800 (LI-COR) secondary antibody for 1 hour at room temperature. The specific signals and the corresponding band intensities were evaluated using an Odyssey Infrared Imaging system and software (LI-COR Bioscience). The primary antibodies used for the Western blot analysis are listed in Table S1 .
5
Quantification of Angiotensin Receptor Proteins
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Extracts of rat tissue were collected using tissue lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) plus 1 mM phenylmethylsulfonyl fluoride. Total protein was quantified using a bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with PBS and Tween 20 containing 5% non-fat dry milk to prevent nonspecific antibody binding. Equal protein loading was determined using specific antibodies for the antigens of GAPDH (CWBiotech, Beijing, China), ACE1, AT1R and AT2R (Abcam, Cambridge, MA, USA), washed, and incubated with an appropriate IRDye800-conjugated secondary antibody (Rockland, Gilbertsville, PA, USA). Specific immunofluorescence bands were detected and analyzed using the Odyssey infrared imaging system and software (LI-COR Biosciences, Lincoln, NE, USA).
6
Western Blot Analysis of RTK Signaling
7
Protein Expression Analysis via Native PAGE and Western Blotting
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Each sample (20 μl) was dissolved in 7.5 μl loading buffer. The samples were applied with a protein marker on a native polyacrylamide gel, which had been equilibrated in a forerun with 0,5× TBE as running buffer for 30 min at 25 mA. The subsequent run with the samples was at 25 mA for 3 h. The blotting onto a PVDF membrane (Millipore Corp., Bedford, MA, USA), which had been previously activated in 99% ethanol, was carried out overnight at 40 V and 80 mA. Immunoblotting was performed with polyclonal antibodies: anti-FAIM2 (1:200 dilution), anti-Trim21 (1:200 dilution) (both from Santa Cruz Biotechnology, Inc.) and anti-actin (1:200 dilution; Abcam, Cambridge, UK). As a secondary antibody, Odyssey 600 anti-rabbit and Odyssey 800 anti-goat (Invitrogen) were used for the quantification of protein expression levels, and signals were obtained using the Odyssey Infrared Imaging System and software (Li-Cor Biosciences, Lincoln, NE, USA).
8
Quantifying Keratinocyte Protein Expression
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Total protein extract of primary keratinocytes was made in a radioimmunoprecipitation assay buffer (RIPA) containing 0.3 M NaCl, 1% sodium desoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Triton-X-100, 20 mM Tris–HCl (pH 8), and 1 mM ethylene diamine tetraacetic acid (EDTA) supplemented with 1 mM phenylmethyl sulfonyl fluoride (PMSF). A total of 25 µg of each protein sample were separated by electrophoresis on 15% SDS–polyacrylamide gels and then transferred on a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Bedford, MA, USA). Immunoblotting was performed with monoclonal rabbit Anti-MHC class I + HLA-A + HLA-B antibody (abcam, Cambridge, UK). Odyssey 680/800 nm secondary conjugates (Li-Cor BioSciences, Lincoln, NE, USA) were used for the quantification of protein expression levels and signals were visualized using the Odyssey Infra-Red Imaging System and software (Li-Cor BioSciences, Lincoln, NE, USA).
9
Western Blot Analysis of Muscle Proteins
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Briefly, muscles were lysed with bead homogenizer (Precelly 24 lysis & homogenization, Bertin technologies) in ice-cold RIPA buffer: 25 mM Tris pH 7.6, 150 mM NaCl, 1 mM Na3VO4, 10 mM NaPyroPO4, 10 mM β-glycerophosphate, 10 mM NaF, 1 mM PMSF, 1X protease inhibitor cocktail (Santa Cruz), 1% NP40, 1% sodium deoxycholate and 0.1% SDS. After quantification by BCA protein assay (Thermo Scientific) equal amounts of muscle homogenates were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore, Billercia, United States) and probed using primary antibodies from the vendors and at the dilutions shown in Supplementary Table 1 with LI-COR IRDye secondary antibodies (LI-COR Inc, Lincoln, United States). Immunoreactive bands were visualized and quantified by densitometry using the Odyssey Infrared Imaging System and software (LI-COR). To control for variations among different western blots and allow for multiple repeats, the absorbance intensity for each band on the western blot was normalized to the absorbance intensity of corresponding band from the control mice from the same western blot, averaged with values obtained from other western blots and plotted as % control. Some blots had multiple controls and the absorbance of the specific band was normalized to the average56 (link). Full length western blots are show in Supplementary Fig. 9 .
10
Western Blot Analysis Protocol
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Western blot analysis was performed as described previously [58 ]. The proteins were electrophoresed under reducing conditions in 12% SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were blocked in 5% BSA and incubated overnight at 4°C with the primary antibody, followed by incubation with the secondary antibody for 1 hour at room temperature. The primary and secondary antibodies used for the Western blot are listed in Table S2 . The specific signals and the corresponding band intensities were evaluated using an Odyssey Infrared Imaging system and software (LI-COR Bioscience, NE, USA).
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