The small intestine of db/db mice was dissected and lysed in a radio immuno precipitation assay (RIPA) buffer (50 mM Tris–HCl (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF) that contained a phosphatase inhibitor cocktail. The lysed cells were then subjected to electrophoresis using sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes. The membranes were reacted with primary antibodies for 3 h and then incubated with the appropriate goat peroxide-conjugated secondary antibodies for 1 h at room temperature. The proteins on the membranes were detected with a chemiluminescent detection kit (Intron Biotechnology) and visualized using the LAS4000 chemiluminescent image analyzer (Fuji, Tokyo, Japan).
Las 4000 chemiluminescent image analyzer
Manufactured by Fujifilm
Sourced in Japan
The LAS 4000 is a chemiluminescent image analyzer manufactured by Fujifilm. It is designed to detect and quantify chemiluminescent signals, a common technique used in molecular biology and biochemistry. The device captures and analyzes images of chemiluminescent samples, providing data on the intensity and distribution of the signals.
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Lab products found in correlation
Anti myc, by Roche (1 mentions)
Anti h2ax, by Cell Signaling Technology (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Anti k8, by Santa Cruz Biotechnology (1 mentions)
Anti par, by Bio-Techne (1 mentions)
Anti chfr, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Anti parp1, by BD (1 mentions)
Anti uhrf1, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Phospho lkb1, by Cell Signaling Technology (1 mentions)
Lumiglo reagent, by Cell Signaling Technology (1 mentions)
Phospho ampk, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
West pico chemiluminescent kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using las 4000 chemiluminescent image analyzer
1
Analyzing Protein Expression in db/db Mice
2
Western Blot Analysis of Protein Expression
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Whole-cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The resolved proteins were transferred to a polyvinylidene fluoride membrane (0.45 μm pore size). The membrane was probed with primary antibodies: anti-FLAG-M2 (1:2,000; Sigma, St. Louis, MO, USA), anti-MYC (1:500; laboratory-made or 1:2,000; Roche), anti-RTA (1:500; laboratory-made), anti-K8 (1:500; laboratory-made), anti-GFP (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-PARP1 (1:1,000; BD Biosciences), anti-PAR (1:500; Trevigen), anti-CHFR (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-UHRF1 (1:500; Santa Cruz Biotechnology), anti-H2AX (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-γH2AX (1:500; Merck Millipore, Billerica, MA, USA) or anti-α-tubulin (1:2,000; Sigma). The membrane was then incubated with the horseradish peroxidase–conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G antibody (1:5000; a secondary antibody; Santa Cruz Biotechnology). The protein bands were detected with enhanced chemiluminescence (ECL) and western blotting detection reagents (ELPIS, Taejeon, Republic of Korea). The protein bands were documented on an LAS-4000 chemiluminescent image analyzer (Fujifilm). The band intensities were calculated in the ImageJ software [72 (link)].
3
Western Blot Analysis of Liver Proteins
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Liver tissue was homogenized using Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, and 1% phenylmethylsulfonyl fluoride (PMSF). The tissue extracts were centrifuged at 12,000 ×g at 4°C for 20 min and the protein concentration of the supernatant was determined using a BioRad protein assay according to the manufacturer's protocol (Bio-Rad Laboratories, CA, USA). Equivalent amounts of protein of each sample were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Shanghai, P.R. China). The membranes were then blocked with 5% skim milk and subsequently incubated with the appropriate primary antibodies liver kinase B (LKB1) (Cat No. 3047), phospho-LKB1 (Cat No. 3482), AMPK (Cat No. 2532), phospho-AMPK (Cat No. 2531), acetyl-CoA carboxylase1 (Cat No. 3676), and phospho-acetyl-CoA carboxylase1 (Cat No. 3661) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), and β-actin (Cat No. SC-47778, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were subsequently reacted with horseradish peroxidase-conjugated secondary antibody (Cat No. 7074S). The immunoreactive bands were visualized by incubation with lumiGLO reagent (Cell Signaling, Beverly, MA, USA) and analyzed using an LAS 4000 chemiluminescent image analyzer (Fuji, Tokyo, Japan).
4
Perilla Extract Modulates UV-Induced DNA Damage
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HaCaT cells were cultured in 6-well plates, treated with Perilla leaf or callus extracts for 12 h, UVB-irradiated, and then incubated for 3 h at 37 °C [14 (link)]. Following incubation, cells were harvested with trypsin-EDTA, resuspended in lysis buffer on ice for 40 min, centrifuged at 13,714× g for 20 min at 4 °C, and protein concentrations in supernatants were determined using a NanoDrop (NanoDrop Lite spectrophotometer, Waltham, MA, USA). Equal amounts of proteins were subjected to SDS-PAGE and then transferred to nitrocellulose membranes, which were reacted with primary antibodies; pCHK1 (Serine 345), γH2AX (both from Cell Signaling, Danvers, MA, USA), CyclinD1, Cell division protein kinase 6 (CDK6), and β-actin (all antibodies from Santa Cruz Bicycles, Santa Cruz, CA, USA) at 4 °C overnight. The following day, membranes were incubated with anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies for 40 min at room temperature. Blots were detected using the West Pico chemiluminescent kit (Thermo, Rockford, IL, USA) and visualized using LAS 4000 chemiluminescent image analyzer (Fuji, Tokyo, Japan).
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