For sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), cells were lysed in lysis buffer (25 mM Tris–HCl [pH 7.6], 150 mM NaCl, 1% NP‐40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktail). Lysates were centrifuged at 15,000 × g for 10 minutes at 4°C to remove debris. For immunoblotting, supernatants were subjected to 10 to 20% gradient SDS‐PAGE. Proteins were transfected onto a polyvinylidene fluoride membrane and probed with anti‐CPT1A (ab128568; Abcam, Cambridge, MA), anti‐β‐actin (MAB1501; EMD Millipore, Billerica, MA) and anti‐GAPDH (ab9485; Abcam) antibodies. This was followed by detection with an LAS‐4000 Mini instrument (GE Healthcare, Chicago, IL). Signal intensities were quantified using ImageJ software (https://imagej.nih.gov/ij/index.html ).
Las 4000 mini instrument
Manufactured by GE Healthcare
The LAS-4000 Mini instrument is a compact fluorescence imaging system designed for small-scale applications. It features a high-resolution CCD camera and multiple excitation and emission filter options to enable the detection and analysis of a variety of fluorescent signals. The core function of the LAS-4000 Mini is to provide a platform for capturing and analyzing fluorescent images.
Automatically generated - may contain errors
Lab products found in correlation
Ab128568, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Rabbit anti β actin, by Abcam (1 mentions)
Imagequant tl image analysis software, by GE Healthcare (1 mentions)
Ecl select western blotting detection system, by GE Healthcare (1 mentions)
Quick start bradford protein assay, by Bio-Rad (1 mentions)
4 protocols using las 4000 mini instrument
1
SDS-PAGE and Immunoblotting for Protein Analysis
2
Protein Detection by Western Blot
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Samples were resolved by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking in Tris-buffered saline containing 0.1% Tween-20 and 5% (w/v) nonfat dry milk, membranes were incubated with the appropriate primary antibodies, followed by the corresponding secondary antibodies. Bands were visualized by enhanced chemiluminescence, and the signals were detected on a LAS4000 mini-instrument (GE Healthcare).
3
Western Blot Protein Lysis and Detection
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Cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 2 mM PMSF, 2 mM EDTA, 2 mM imidazole, and 10 mM NaF, with or without 5 mM N-ethylmaleimide and 7.5 mM cysteine. Lysates were clarified by centrifugation at 20,000 × g for 20 min at 4°C. The proteins in the lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in Tris-buffered saline containing 0.1% Tween-20 and 5% (wt/vol) nonfat dry milk, the membrane was incubated with the appropriate primary antibodies, followed by appropriate secondary antibodies (see Table S5 ). The membranes were visualized using enhanced chemiluminescence and a LAS4000mini instrument (GE Healthcare).
4
Cerebellar and Forebrain Protein Analysis
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Cerebellar and forebrain homogenates were prepared from 8-week-old mice. The brains were homogenized with homogenate buffer containing 0.32 M sucrose, 1 mM NaHCO3, 1 mM MgCl2, 0.5 mM CaCl2, and protease inhibitors using a Teflon-glass homogenizer, followed by centrifugation at 710 × g for 10 min at 4°C, as described previously 54 . Proteins were quantified using the Quick Start Bradford Protein Assay (Bio-Rad). Equal amounts of protein were separated by SDS-PAGE, transferred to a PVDF membrane, and incubated with rabbit anti-pan NRXN (0.42 μg/ml) 55 and rabbit anti-β-actin (Abcam) (1:2000) antibodies, followed by incubation with a horseradish-peroxidase-conjugated secondary antibody, respectively. Immunoreactive proteins were visualized using the ECL Select Western Blotting Detection System (GE Healthcare). Signals were detected on a Las-4000 mini instrument (GE Healthcare) and quantified using the ImageQuant TL image analysis software (GE Healthcare).
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