Western blotting was performed as previously described [34 (link)]. The cells were homogenized directly in the wells, using RIPA buffer and a rubber policeman, followed by brief sonication (10× 1-second pulses of 20% max; Sonics Vibra-Cell VCX130; Sonics & Materials, Newtown, Conn., USA). Samples were resolved by 4-12% SDS PAGE (Invitrogen) and transferred to PVDF via a semi-dry blotting apparatus (Bio-Rad). Membranes were incubated with primary antibodies and the appropriate fluorescently tagged secondary antibody under the conditions listed in online supplementary table 2. Immunoreactive bands were visualized using the Odyssey Clx infrared scanner (Licor Biosciences), and analysis was performed on the Image Studio software package (Licor Biosciences).
Image studio software package
Manufactured by LI COR
Sourced in United States
Image Studio software is a comprehensive analysis and quantification tool for western blots and other fluorescence-based imaging applications. It provides intuitive image processing, analysis, and data management capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Vibra cell vcx130, by Sonics (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Odyssey clx infrared scanner, by LI COR (1 mentions)
Semi dry blotting apparatus, by Bio-Rad (1 mentions)
Apyrase, by Merck Group (1 mentions)
Clx scanner, by LI COR (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Odyssey fc, by LI COR (1 mentions)
Rabbit polyclonal anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
Blotting buffer, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
C digit scanner, by LI COR (1 mentions)
Westernbright sirius reagent, by Advansta (1 mentions)
4 protocols using image studio software package
1
Quantitative Western Blotting Protocol
2
Ubiquitin Discharge Assay Protocol
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E2 enzymes were charged with ubiquitin under the following conditions: 1 μM E1, 1 μM UbiquitinAtto, 3 mM ATP, 4 μM E2 (reaction buffer: 50 mM Hepes, pH 7.5, 150 mM NaCl, 20 mM MgCl2, and 5 mM CaCl2). The reactions were incubated at 25°C for 20 min, followed by a 5-min incubation with 1U of apyrase (Sigma-Aldrich). Discharge was initiated by addition of L-lysine, at a final concentration of 20 mM, and E3, at a final concentration of 4 μM. Discharge assays were incubated at 25°C for up to 60 min, the samples were quenched by addition of SDS sample buffer and flash-freezing on dry ice at the described time intervals, and resolved by SDS–PAGE. For quantification, the gels were scanned with a LICOR CLx scanner, and the bands for E2–UbAtto were integrated using the ImageStudio software package (LI-COR). Experiments were performed in duplicate (UBE2E2) or triplicate (UBE2D and UBE2E1). The scans were converted to greyscale.
3
Western Blot Analysis of IL-37 Expression
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The protein concentrations of the cell lysate were determined using a DC Protein Assay Kit (Bio-Rad). To detect the expression of IL-37 in IL-37sgRNA and TFneg cells, 50 μg total protein from cell lysates was resolved in 12% Bis/Tris gels (Novex, Life Technologies) in NuPage running buffer (Novex, Life Technologies) and transferred to nitrocellulose membranes in blotting buffer (Bio-Rad). After blocking in 5% bovine serum albumin (BSA, Carl Roth GmbH, Karlsruhe, Germany), nitrocellulose membranes were probed overnight at 4 °C using 3 µg/ml rabbit polyclonal anti-IL-37b (Novus Biologicals, Cambridge, UK). Rabbit polyclonal anti-GAPDH (Santa Cruz Biotechnology, Dallas, Texas) at a 1:15,000 dilution was used as a loading control. Blots were then incubated with a horseradish peroxidase-conjugated secondary anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) and visualized using a Luminata Forte Western HRP substrate (Merck Millipore, Billerica, MA, USA). The membranes were exposed to chemiluminescence using an Odyssey Fc (LI-COR Biosciences, Lincoln, NE, USA). Densitometric analysis was performed using version 3.1 of the Image Studio software package (LI-COR). The calculations were performed based on density of the IL-37 and GAPDH bands, respectively, as detected by the software, and the results were normalized based on IL-37/GAPDH.
4
Cytokine Profile Analysis of Conditioned Media
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Conditioned media was analyzed using the Proteome Pro ler Human Cytokine Array (R&D Systems) as directed by the manufacturer. The arrays were developed using WesternBright Sirius reagent (Advansta, San Jose, California, USA) on a C-Digit scanner (LI-COR, Lincoln, Nebraska, USA) and signal densities were determined using Image Studio software package (LI-COR). Data were normalized to CSDS strain pro le for each paired ex run.
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