Whole-cell lysates were prepared by resuspending cells in RIPA buffer containing 5× cOmplete Mini Protease Inhibitor Cocktail (Sigma) and homogenised with a 19-gauge syringe. Equal amounts of protein samples were boiled in Laemmli buffer, resolved by a Mini-PROTEAN TGX Precast Gel (Bio-Rad), transferred to a nitrocellulose membrane by a Trans-Blot Turbo Transfer System (Bio-Rad), incubated with primary antibodies against EDNRA (Abcam; ab117521; 1:2500 dilution; 48 kDa and glycosylated EDNRA 60–90 kDa47 (link)) and ACTB (Cell Signalling; #8457; 1:1000 dilution; 45 kDa), and subsequently a goat anti-rabbit horseradish peroxidase secondary antibody (Pierce; 1:1000 dilution). The membrane was incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) before being imaged with a Molecular Imager ChemiDo XRS+System (Bio-Rad).
Molecular imager chemido xrs system
Manufactured by Bio-Rad
Sourced in United States
The Molecular Imager ChemiDo XRS System is a high-performance imaging system designed for chemiluminescent and fluorescent applications in molecular biology and biochemistry. It features a high-resolution camera, adjustable lighting, and data analysis software to capture and analyze images of protein gels, Western blots, and other molecular assays.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Ab117521, by Abcam (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse igg antibody, by Proteintech (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cell lysis reagent, by Beyotime (1 mentions)
Chemiluminescence detection kit, by Bio-Rad (1 mentions)
Polyvinylidene fluoride pvdf membrane, by GE Healthcare (1 mentions)
Anti actin, by Proteintech (1 mentions)
3 protocols using molecular imager chemido xrs system
1
Western Blot Analysis of EDNRA Protein
2
Western Blot Analysis of Protein Expressions
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Whole body (50), midgut (50) and salivary gland (100) samples of adults (female / male ratio = 1:1) were collected and lysed with TRIzol reagent (ThermoFisher, USA) or Cell Lysis reagent (Beyotime, China) for protein extraction according to the manufacturer’s protocols. After adding 6× SDS loading buffer, 50 μg protein samples were boiled for 10 min. The proteins were separated by 8–12% SDS-PAGE and transferred onto PVDF membranes. Blots were probed with the following antibodies: anti-LsTUB (1:1000 dilution), anti-RSV CP (1:1000 dilution), anti-RSV NS3 (1:500 dilution), or anti-GAPDH (1:2000 dilution). Immuno-reactive bands were detected using a goat anti-rabbit/goat anti-mouse IgG-conjugated HRP antibody and a goat anti-mouse IgG-conjugated HRP antibody (Proteintech, USA) at a 1:5000 dilution. Western blots were imaged with a Chemiluminescence Detection Kit (Bio-Rad, Hercules, CA, USA) and the Molecular Imager ChemiDo XRS System (Bio-Rad). Triplicate samples were performed for each experiment and the relative intensities of protein expressions were calculated using Image lab 5.2.1 software (Bio-Rad).
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted from SPBHs according to our published protocol with minor modifications (Liu et al., 2015 (link)): 0.02 g of SBPH was ground in liquid nitrogen to powder and 200 μl of 50 mM Tris‐HCl was added. After vortexing and vertical shaking for 1 hr at 4 ℃, the mixture was centrifuged at 4 ℃ for 20 min. Then we took 80 μl of supernatant and mixed with 20 μl of 5× sodium dodecyl sulphate (SDS) loading buffer, then subsequently boiled the mixture for 10 min. The denatured proteins were separated by 4%–12% SDS‐polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare Life Sciences) by electrophoresis at 150 mA for 2 hr. Proteins were then probed with anti‐RBSDV P10 (1:2,500 dilution) or anti‐actin (1:4,000 dilution, 66,008; Proteintech). Immunoreactive bands were detected using a goat anti‐mouse IgG‐conjugated horseradish peroxidase antibody (5220‐0341; SeraCare) at 1:5,000 dilution. Western blots were imaged with a Super ECL Western Blotting Detection Kit (YTHX) and Molecular Imager ChemiDo XRS System (Bio‐Rad). Each western blot was repeated three times.
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