Cells were lysed in radioimmunoprecipitation assay buffer (Tris-HCl, pH 7.4, 50 mM; NaCl, 150 mM; NP-40 1%; sodium deoxycholate, 0.5%; sodium dodecyl sulfate, 0.1%; EDTA, 2 mM; sodium fluoride, 50 mM) supplemented with protease and phosphatase inhibitor cocktails (Roche, Mississauga, Canada). Protein concentrations were determined by Bradford protein assay (Bio-Rad, Mississauga, Canada). Loading buffer was added to 30 μg of protein lysate and resolved by SDS–PAGE. The protein was then transferred onto a polyvinylidene difluoride membrane (Millipore, Toronto, Canada) and probed using antibodies for PI4KIIIβ (BD Biosciences 611817; Mississauga, Canada), pan-actin (Cell Signaling Technology 4968), tubulin (1:1000, Cell Signaling Technology 3873; Whitby, Canada), mouse α-FAK (1:200 Thermo Fisher #396500), rabbit α-Phospho-FAK (1:200 ThermoFisher #44-626), as well as anti-rabbit HRP-linked (Cell Signaling Technology catalogue no. 7074). Bands were detected with a MicroChemi chemiluminescent system (DNR Bio-Imaging Systems, Toronto, Canada) and intensities were quantified by densitometry using GelQuant (DNR Bio-Imaging Systems).
Microchemi chemiluminescent system
Manufactured by DNR Bio-Imaging Systems
Sourced in Canada, Israel
The MicroChemi chemiluminescent system is a compact and versatile imaging device designed for detecting and analyzing chemiluminescent signals. It utilizes a high-performance CCD camera to capture digital images of chemiluminescent samples, enabling sensitive detection and quantification of various biomolecules and biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Pan actin, by Cell Signaling Technology (1 mentions)
Pi4kiiiβ, by BD (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Amersham ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Any kd mini protean tgx precast polyacrylamide gels, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Gapdh, by GeneTex (1 mentions)
2 protocols using microchemi chemiluminescent system
1
Immunoblotting with Cellular Lysate
2
Western Blot Analysis of Cartilage Proteins
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Constructs were frozen in liquid nitrogen, ground into fine particles, resuspended in 30–50 mL Laemmli sample buffer, sonicated on ice, and then boiled for 10 min, after which the DNA concentration of the samples was determined. Proteins standardized by DNA content were separated on 7.5% or Any kD Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad) and transferred to PVDF membranes (Bio-Rad). The membranes were probed with the following primary antibodies: rabbit polyclonal type I (1:1000, Proteintech), type II collagens (1:1000, Santa Cruz Biotechnology), α-Tubulin (1:1000, MBL), GAPDH (1:1000, GeneTex), and goat polyclonal aggrecan (1:1000, R&D Systems) antibodies. After multiple washes, the membranes were incubated with HRP-conjugated anti-rabbit or anti-goat IgG (eBioscience). After multiple washes, the membranes were treated with the Amersham ECL Prime Western Blotting Detection Reagent (Cytiva). Images of bound antibodies were digitally captured using a MicroChemi chemiluminescent system (DNR Bio-Imaging Systems, Israel), and band intensities were quantified using GelQuant Analysis Software (DNR Bio-Imaging Systems).
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