Tissue samples were subjected to immunoblotting analysis as described previously [15 (link)]. Briefly, liver tissue was homogenized in lysis buffer and centrifuged at 13,000 rpm, 4°C for 15 minutes. The total concentration of protein was measured using the Bradford protein assay dye (Bio-Rad Laboratories, Hercules, CA, USA). The whole lysate was mixed with 5× sample buffer and denatured at 95°C for 6 minutes. The total proteins were separated by SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (GE Healthcare BioSciences Co., Piscataway, NJ, USA). After protein blocking, the membranes were incubated with anti-phospho-nuclear factor kappa B (p-NF-κB, 1:1,000, #3033, Cell Signaling Technology), anti-total-nuclear factor kappa B (t-NF-κB, 1:1,000, #8243, Cell Signaling Technology), anti-acyl-CoA oxidase 1 (ACOX1; 1:1,000, sc-98499, Santa Cruz Biotechnology Inc.), and anti-β-actin (1:3,000, A5326, Sigma-Aldrich) antibodies. The blots were reacted with peroxidase-conjugated secondary antibodies (Vector Laboratories Inc., Burlingame, CA, USA), and the positive immunoreactive protein bands were detected using LAS-3000 film (FUJIFILM Corporation, Tokyo, Japan). All protein levels were normalized to β-actin.
Las 3000 film
Manufactured by Fujifilm
Sourced in Japan
The LAS-3000 film is a lab equipment product by Fujifilm. It is designed for the capture and analysis of images. The device utilizes a charge-coupled device (CCD) sensor to capture high-quality images.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated secondary antibody, by Vector Laboratories (2 mentions)
Bradford protein assay dye, by Bio-Rad (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Ecl reagent, by PerkinElmer (1 mentions)
Pvdf membrane, by PerkinElmer (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti p src, by Cell Signaling Technology (1 mentions)
Anti cox4i1, by Cusabio (1 mentions)
Anti p fyn, by Santa Cruz Biotechnology (1 mentions)
Anti tfam, by Abcam (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Anti nrf1, by Abcam (1 mentions)
Anti pgc 1α, by Abcam (1 mentions)
4 protocols using las 3000 film
1
Immunoblotting of Liver Proteins
2
Cytokine Profiling of Thrombin-Treated Cells
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Supernatants collected from NP cells were then removed and clarified with a 20,000 g spin (10 s at 4 °C), and applied to each of two separate cytokine array membranes. Membranes were processed following the manufacturer’s instructions. Chemi-luminescence was detected using LAS-3000 film (Fuji; Tokyo, Japan). Manual background subtraction was performed prior to quantitation, and “fold change” was calculated as the difference between the average values for both cytokine spots on thrombin-treated versus untreated arrays.
3
Antroquinonol's Molecular Effects
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The cells (5 x 105) were seeded in a 6 cm dish, grown until 80% confluent, and then incubated with various concentrations of antroquinonol for 24 h. Cells were collected and lysed with RIPA buffer. Protein samples were loaded and separated with SDS-PAGE then transferred to the PVDF membrane (PerkinElmer, Turku, Finland). The membranes were incubated with appropriate primary antibodies at 4°C overnight. The membranes were washed 3 times with TBST, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. Finally, the membranes were exposed to ECL reagents (PerkinElmer) for 1 min and the results were analyzed with LAS-3000 film (Fujifilm, Tokyo, Japan). Beta actin was used as an internal control.
4
Kidney Protein Extraction and Western Blot Analysis
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Whole kidney protein was extracted with lysis buffer. After centrifugation (13,000 rpm, 4 °C, 15 min), the lysate was mixed with 5× sample buffer and heated at 95 °C for 6 min. Total protein concentrations were measured using Bradford methods (BioRad Laboratories, Hercules, CA, USA). The lysate was subjected to SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes were incubated overnight at 4 °C with the primary antibodies: anti-p-Src (1:1000; Cell Signaling Technology), anti-p-Fyn (1:1000; Santa Cruz Biotechnology), anti-PGC-1α (1:1000; Abcam), anti-TFAM (1:1000; Abcam), anti-NRF1 (1:1000; Abcam), anti-Cox4i1 (1:1000; Cusabio Biotech Co., Baltimore, MD, USA), anti-β-actin (1:1000), and anti-heat shock 70 kDa protein 8 (HSC70, 1:1000; Santa Cruz Biotechnology). The blots were reacted with peroxidase-conjugated secondary antibodies (Vector Laboratories, Inc., Burlingame, CA, USA), followed by an enhanced chemiluminescent sensitive plus reaction (BioFX Laboratories, Inc., Owings Mills, MD, USA). The positive immunoreactive protein bands were detected by LAS-3000 film (FUJIFILM Corporation, Tokyo, Japan). Each blot density was normalized to β-actin or heat shock 70 kDa protein 8 and compared with that of each control.
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