Proteins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were transferred onto nitrocellulose membranes. After blocking in 5% skim milk powder in Tris-buffered saline, pH 7.5, 0.1% Tween-20 for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. The membranes were then incubated with secondary antibody and visualized using an enhanced chemiluminescence detection kit (Millipore) on a LAS500 system (GE Healthcare). Western blots were analyzed with Imagequant (GE Healthcare).
Las500 system
Manufactured by GE Healthcare
Sourced in United States
The LAS500 system is a laboratory equipment designed for the analysis and measurement of various samples. It provides precise and reliable data on the properties and characteristics of the analyzed materials.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using las500 system
1
Western Blot Protein Detection
2
Western Blot Transfer and Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes. After blocking with blocking buffer (Tris-buffered saline [TBS], pH 7.5, with 0.1% Tween-20 [TBST] for phospho-conjugated antibodies or phosphate-buffered saline [PBS] with 0.1% Tween-20 [PBST] containing 5% skim milk powder) for 1 h at room temperature, the membranes were incubated overnight at 4°C with the primary antibody. After incubation with secondary antibodies conjugated with HRP, the proteins were visualized using an ECL detection kit (Millipore) and the images acquired with a LAS500 system (GE Healthcare).
3
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline-Tween 20 (TBS-T; 20 mM Tris, 150 mM NaCl, 0.2% Tween 20, pH 7.6), probed with appropriate primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. Proteins of interest were visualized by chemiluminescence using a LAS500 system (GE Healthcare, Chicago, IL, USA). Densitometric quantification was performed in unsaturated images using ImageJ (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Hepatic and VAT Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hepatic and VAT samples were collected and washed with PBS and disrupted in lysis buffer (0.25 M Tris-HCl, 125 mM NaCl, 1% TritonX-100, 0.5% SDS, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 2 mM Na3VO4, 10 mM βglycerophosphate, 2.5 mM sodium pyrophosphate, 10 mM PMSF, 40 µL of protease inhibitor) using the TissueLyser system (Quiagen, Hilden, Germany). The bicinchoninic acid (BCA) Protein Assay Kit was carried out on the supernatant (14,000 rpm for 20 min at 4 °C, followed by the addition of Laemmeli buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.01% bromophenol blue). Tissue samples (20 µg) were loaded onto SDS-PAGE and electroblotted into polyvinylidene difluoride (PVDF) membrane (Advansta, San Jose, CA, USA). Tris-buffered saline-tween (TBS-T) 0.01% and bovine serum albumin (BSA) 5% were used to block the membranes, which were then incubated with primary (overnight, 4 °C) and secondary antibodies (2 h RT), following the dilutions listed in the Supplementary Table S1 . The proteins of interest were detected using enhanced chemiluminescence (ECL) substrate with the LAS 500 system (GE Healthcare, Chicago, IL, USA). The bands of interest were quantified with Image Quant 5.0 software (Molecular Dynamics). The results were expressed as a percentage of control and normalized for the loading control (calnexin, 83 kDa).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!