Washed platelets were lysed, separated by SDS–polyacrylamide gel electrophoresis, and blotted onto polyvinylidene difluoride membranes. Membranes were incubated with the respective antibodies against glyceraldehyde-3-phosphate dehydrogenase (1:1000; Sigma-Aldrich) and myosin IIA (1:200; Sigma-Aldrich). Horseradish peroxidase (HRP)–conjugated secondary antibodies and enhanced chemiluminescence solution (Bio-Rad Clarity Western ECL Substrate) were used for visualization on an Amersham Imager 680 (GE Healthcare).
Clarity western ecl substrate
Manufactured by GE Healthcare
Sourced in United Kingdom
Clarity Western ECL substrate is a chemiluminescent detection reagent used in Western blotting to visualize proteins. It generates a luminescent signal upon interaction with the enzyme label on the secondary antibody, allowing for the detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one 1d image analysis software, by Bio-Rad (2 mentions)
Nupage novex bis tris midi gel 15 or 26 wells, by Thermo Fisher Scientific (2 mentions)
Amersham imager 680, by GE Healthcare (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Merck Group (1 mentions)
Myosin iia, by Merck Group (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Ab21058, by Abcam (1 mentions)
Fusion tx7, by Thermo Fisher Scientific (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Ripa lysis buffer, by G Biosciences (1 mentions)
5 protocols using clarity western ecl substrate
1
Protein Expression Analysis by Western Blot
2
Microdystrophin Protein Quantification in mdx Mouse
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Total protein was isolated from quadriceps and hearts of mdxGT and mdxRGT animals using RIPA buffer (Millipore, #20-188) containing protease inhibitors (Roche, #11836153001) (Roche, #05892970001). Proteins were transferred to nitrocellulose membranes and probed for microdystrophin using the NCL-DYSB primary antibody (Leica, 6052319, 1:50) and a horseradish peroxidase (HRP)-linked anti-mouse insulin growth factor (IgG) secondary antibody (Jackson Labs, #715-035-150, 1:4,000). Signal detection was performed using the Clarity Western ECL substrate (GE Life Sciences, #RPN2236) and measured by digital imaging using the ChemiDoc MP system (Bio-Rad). Quantification of band signal intensities normalized to β-tubulin (Abcam, Ab21058, 1:5,000) was performed with Image Lab Software (Bio-Rad). Relative protein levels were evaluated using a one-way ANOVA with Prism software (GraphPad Prism 8).
3
Western Blot Protein Analysis Protocol
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Equal amounts of total protein extract (30 μg) were electrophoretically separated using SDS–PAGE in 4–12% Bis–Tris gels (NuPAGE® Novex Bis-tris midi gel 15 or 26 wells, Life Technologies, Carlsbad, USA) and transferred to nitrocellulose membranes. Blocked membranes (5% non-fat dry milk in TBS/0.1% Tween-20) were incubated with the primary antibodies (Table 1 ) overnight at 4 °C, and washed three times with TBS/0.1% Tween-20 (T-BST) for 10 min. Membranes were then labelled with secondary anti-IgG-HRP antibodies raised against each corresponding primary antibody. After three washes with T-BST, the membranes were incubated with ECL chemiluminescent reagent (Clarity Western ECL substrate; GE Healthcare, Little Chalfont, UK) according to the instructions of the supplier. Peroxidase activity was detected with the camera system Fusion TX7 (Fisher Scientific). Normalization was done by densitometry analysis with the Quantity One 1D image analysis software (version 4.4; Biorad, Hercules, CA, USA). The optical densities were normalized with respect to a housekeeping protein (GAPDH, actin or tubulin). A partition ratio was calculated and normalized with respect to the sample with the highest value defined as one.
4
Western Blot Analysis of Proteins
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Protein was extracted from Scs by resuspending the cells in RIPA Lysis buffer (G Biosciences) with 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail followed by freeze-thaw in liquid nitrogen. For the testis, the tissue was homogenized in RIPA Lysis buffer (with 1× protease and phosphatase inhibitor cocktail). Protein was quantified using Bradford solution,75 and 20–30 μg total protein was run on 10% or 12% resolving SDS-PAGE gel at 100 mV. The protein was transferred onto PVDF membranes and incubated with blocking solution (5% skimmed non-fat milk in 1× Tris buffered saline with 0.1% Tween 20 detergent or 1XTBST) for 1–2 h at room temperature. The membrane was briefly washed with 1× TBST and incubated overnight with the primary antibody dissolved in 1× TBST with or without 1% BSA. The next day the membrane was washed thrice with 1× TBST (5 min each at RT under rapid shaking condition) and incubated for 1 h with the horseradish peroxidase (HRP)-labeled secondary antibody in 1× TBST. The membrane was then developed using BioRad Clarity Western ECL substrate and imaged in a chemi-doc (ImageQuant Las 500, GE Healthcare). The protein bands obtained were quantified using ImageJ. β-actin was used as loading control. Primary antibody dilutions are listed in Table S5 .
5
Quantitative Western Blot Analysis
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Total protein concentrations were determined using the BCA kit (Pierce). Equal amounts of total protein extract (30 µg) were electrophoretically separated using SDS-PAGE in 4-12% Bis-Tris gels (NuPAGE ® Novex Bis-tris midi gel 15 or 26 wells, Life Technologies, Carlsbad, USA) and transferred to nitrocellulose membranes. Blocked membranes (5% non-fat dry milk in TBS-0.1% Tween-20) were incubated with primary antibodies overnight at 4 °C and washed three times with TBS-0.1% Tween-20 (T-BST) for 10 min. Membranes were then labeled with secondary IgG-HRP antibodies raised against each corresponding primary antibody. After three washes with T-BST, the membranes were incubated with ECL chemiluminescent reagent (Clarity Western ECL substrate; GE Healthcare, Little Chalfont, UK) according to the instructions of the supplier. Peroxydase activity was detected with a Fusion TX7 camera system (Fisher Scientific). Quantification was made by densitometric analysis with the Quantity One 1D image analysis software (version 4.4; Biorad, Hercules, CA, USA). The optical densities were normalized with respect to a "standard protein" (GAPDH/actin). For each sample, a partition ratio was calculated and normalized with respect to the sample with the highest value defined as 1.
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