Retina tissues and cells were lysed using a lysis buffer (125 mM HCl [pH 6.8], 4% SDS, 20% glycerol, urea 0.36 g/mL, 20 mM DTT, 1% proteinase inhibitor cocktail), and the proteins were resuspended in SDS-PAGE loading buffer. Equal amounts of samples were loaded in each lane. The proteins were separated in 12% polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were then probed with primary antibodies against Mbd2 (GenBank: 17191) (#ab188474; Abcam, Cambridge, MA, US), caspase3/cleaved caspase3 (#9662; CST, Danvers, MA, US), Traf3 (GenBank: 22031) (#ab36988; Abcam), and β-tubulin (#10094-1-AP; Proteintech, Rosemont, IL, US), followed by incubation with species-specific horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, SA00001-2; Proteintech), and visualized by enhanced chemiluminescence (Millipore; WBKLSO500). Finally, the images were captured by Tanon 5200 Multi. The target proteins were quantified by Photoshop software and normalized by internal control β-tubulin to get relative expression levels.
Wbklso500
Manufactured by Merck Group
Sourced in United States, Germany
The WBKLSO500 is a laboratory equipment product manufactured by the Merck Group. It is a high-performance device designed for use in various scientific and research applications. The core function of the WBKLSO500 is to perform precise measurements and analysis of samples.
Automatically generated - may contain errors
4 protocols using wbklso500
1
Western Blot Analysis of Retinal Proteins
2
Proteomic Analysis of Mouse Cortex
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Briefly, the cortex of mice was dissected and homogenized with RIPA buffer (10 mM Tris-HCl, 137 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, pH 7.4) containing protease inhibitor. After centrifugation at 12,000 rpm for 20 min, the collected protein samples were subjected to SDS-PAGE. The transferred blots were then incubated with primary antibodies at 4°C overnight and HRP-conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature for 2 h, then detected with enhanced chemiluminescence (Millipore, Cat# WBKLSO500). ImageJ software was applied to determine the intensity of each indicated blotting bands.
3
Western Blotting of Cell Proteins
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Western blotting was performed as described previously.42 (link) In brief, proteins were first extracted from cells or retinal tissues. Total protein (30 μg) was then loaded and separated on 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes. Blots were incubated with a primary antibody against Mbd2 (ab188474; Abcam), Atf1 (11946-1-AP; Proteintech, Rosemont, IL), caspase3/cleaved caspase3 (#9662; CST, Danvers, MA), and β-tubulin (#10094-1-AP; Proteintech) at 4°C overnight. The following morning, membranes were washed and incubated with a species-specific horseradish peroxidase-conjugated secondary antibody, Goat Anti-Rabbit IgG (SA00001-2; Proteintech), at RT for 1 h. Positive binding was detected by an enhanced chemiluminescence kit (WBKLSO500; Millipore, Burlington, MA) and captured by a Tanon 5200 multi. Protein levels were normalized to β-tubulin and were compared with controls.
4
Western Blot Analysis of Retinal Proteins
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Proteins were obtained from RGCs and the retinas of mice. Following cell lysis, lysates underwent ultrasonic homogenization and centrifugation and were then denatured by boiling. Protein concentrations were then measured by a NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific, Waltham MA, USA). Equal amounts (30 µg) of proteins were then loaded into each well and separated by 10% or 12% SDS-PAGE. Separated proteins were then transferred onto polyvinylidene difluoride membranes. The blots were then probed with primary antibodies against caspase-3 (1:1000), cleaved caspase-3 (1:1000), Wnt8a (1:2000), and β-tubulin (1:1000). This was followed by incubation with appropriate secondary antibodies (Goat Anti-Rabbit IgG (H + L) HRP [1:5000] or Goat Anti-Mouse IgG (H + L) HRP [1:5000]). Positive antibody binding was visualized by enhanced chemiluminescence (WBKLSO500; Millipore, Merck KGaA, Darmstadt, Germany) and a Tanon-5200Multi system (Tanon, Shanghai, China). Target protein bands were then quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA), and β-tubulin was used as an internal control to normalize the expression levels.
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