Ad libitum–fed plasma apoA-IV protein levels were determined via Western blot analysis as described previously (24 (link)). Briefly, 20 µL 2× SDS sample buffer was added to each sample, boiled for 5 min, and then loaded onto a 10% Tris-HCl gel, with a 4% stacking gel (Bio-Rad Laboratories, Hercules, CA). Gels were run at a constant voltage (80 V) until the protein standards were well separated. Proteins were then transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories) for 1 h at a constant current of 350 mA. After nonspecific binding sites on the membranes were blocked for 1 h with a 5% solution of nonfat milk in Tris-buffered saline with 0.1% Tween (TBS-T), membranes were then incubated with goat anti-rat/mouse apoA-IV antibodies diluted 1:12,000 overnight at 4°C. After incubation, the blots were subsequently washed with nonfat milk in TBS-T, and then incubated with either horseradish peroxidase–conjugated goat anti-rabbit antibodies or with horseradish peroxidase–conjugated rabbit anti-goat antibodies (Dako, Glostrup, Denmark) diluted 1:20,000 with 2.5% nonfat milk in TBS-T at room temperature for 30 min. Detection was achieved by using the enhanced chemiluminescence system (ECL Western Blotting Detection Reagents, Amersham Biosciences, Buckinghamshire, U.K.), and X-OMAT AR films (Kodak) were used for development and visualization of the membranes.
Stacking gel
Manufactured by Bio-Rad
Sourced in United Kingdom, United States
The 4% stacking gel is a type of polyacrylamide gel used in electrophoresis techniques. It is designed to concentrate analytes at the interface between the stacking and resolving gels, improving the resolution and separation of samples during electrophoretic analysis.
Automatically generated - may contain errors
Lab products found in correlation
X omat ar film, by Kodak (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit antibody, by Agilent Technologies (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Ecl western blotting detection reagents, by Kodak (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg or anti mouse igg, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid method, by Merck Group (1 mentions)
Calsequestrin, by Abcam (1 mentions)
Cuznsod, by Enzo Life Sciences (1 mentions)
Polyacrylamide gel, by Bio-Rad (1 mentions)
Mega 900 uv vis spectrophotometer, by Scinco (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
4 protocols using stacking gel
1
Plasma ApoA-IV Protein Quantification
2
Western Blot Analysis of Muscle Proteins
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Muscles were ground in a mortar and pestle under liquid nitrogen, and frozen muscle powder was placed into Radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, and protease inhibitors. Samples were homogenized on ice and centrifuged at 10 000 g for 10 min at 4°C. Protein content of samples was determined using the bicinchoninic acid method (Sigma‐Aldrich, Poole, UK). For assessment of specific proteins in muscle, 20 mg of total protein was applied to a 4–20% mini‐PROTEAN TGX precast gel with a 4% stacking gel (Bio‐Rad Laboratories Ltd, Hemel Hempstead, UK). The separated proteins were transferred onto nitrocellulose membranes by western blotting. Membranes were probed using antibodies against calstabin, serca1, calsequestrin (Abcam, Cambridge, UK), DHPR, RYR (Thermo Scientific, USA), calcineurin, nuclear factor of activated T‐cells (NFAT), calpain (Cell Signaling, Hitchin, UK), and CuZnSod (Enzo, Farmingdale, NY, USA). Horseradish peroxidase conjugated anti‐rabbit IgG or anti‐mouse IgG (Cell Signaling) was used as secondary antibody. Peroxidase activity was detected using an ECL Plus substrate (Amersham International Cardiff, UK), and band intensities were analysed using Quantity One Software (Bio‐Rad Laboratories Ltd). All protein contents were normalized to protein levels determined by the ponceau stain.
3
Biomolecule Immobilization on FPNPs
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The possibility of directly immobilizing biomolecules on FPNPs was tested by their treatment with BSA (at the desired concentration in DI water) for 2 h at 25 °C with gentle mixing. Following immobilization of the BSA, the FPNPs were immediately immersed in DI water and incubated for 4 h to hydrolyze any unreacted NHS esters. After this time, the un-immobilized BSA was removed by centrifugation at 4000g for 30 min. TEM was employed to visualize the BSA-immobilized FPNPs. Electrophoresis, i.e., SDS-PAGE, was performed to assess the conjugation process. Electrophoresis was carried out by regular SDS-PAGE with 12% polyacrylamide gel and 5% stacking gel according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). UV-vis spectra were recorded on a Mega-900 UV-vis spectrophotometer (SCINCO, Seoul, Korea).
4
SDS-PAGE Analysis of Protein Samples
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The protein samples were dissolved in Milli‐Q water at 4 mg mL−1, and then diluted with 2× Laemmli buffer (Bio‐Rad, USA) with the ratio of 1:1 to a final protein concentration of 2 mg mL−1. The diluted protein samples were heated at 100 °C for 5 min (non‐reducing condition); mixed with 10% DL‐Dithiothreitol (1 m ) to Laemmli buffer and heated at 100 °C for 5 min (reducing condition). Afterward, the protein marker (10 µL) and the protein samples (10 µL) were loaded in each well on the gel (12% separating gel and 5% stacking gel, from Bio‐Rad Laboratories Inc.) at 200 V for 30–40 min. The running buffer for electrophoresis (1 L) contained 3 g Tris, 14.4 g Gly, and 1 g SDS, then the pH was adjusted to 8.3. The gels were stained with Coomassie Blue Fast Staining Solution (Beyotime Institute of Biotechnology Co., Ltd., China). Afterward, the gels were destained using distilled water for 24 h. The sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) images were analyzed using the gel imaging system (ChemiDoc XRS+, Bio‐Rad, USA).
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