Protein extracts were made in RIPA buffer and quantitated by BCA assay and diluted to equal concentrations. Polyacrylamide gel electrophoresis was performed on NuPAGE Novex gradient gels (Thermo Fisher) followed by wet transfer to nitrocellulose membranes. Blocking was briefly performed with 5% non-fat milk in PBST and primary antibody was incubated overnight at 4°C in 5% milk in PBST, then with HRP-conjugated secondary antibody (Cell Signaling) at room temperature for 1 hour followed by washing, then developed with ECL pico or femto (Thermo Fisher). For gene expression assays total RNA was isolated with All-prep (QIAGEN), DNase-treated, then reverse-transcribed with Superscript III (Invitrogen). qPCR was performed with 2x KAPA SYBR Fast Master Mix on a QuantStudio Flex 6 (Applied Biosystems).
Novex nupage gradient gels
Manufactured by Thermo Fisher Scientific
Sourced in United States
Novex NuPAGE gradient gels are precast polyacrylamide gels used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins. They are available in various gradient percentages and well configurations to accommodate a range of protein sample sizes and separation needs.
Automatically generated - may contain errors
Lab products found in correlation
γ tubulin antibody, by Merck Group (3 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Goat anti rabbit igg, by Merck Group (2 mentions)
Ncl hamlet, by Leica (2 mentions)
Alexa fluor 680 goat anti mouse, by LI COR (2 mentions)
Quantstudio 6 flex, by Thermo Fisher Scientific (1 mentions)
Ecl pico or femto, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Hrp labeled goat anti mouse igg, by Merck Group (1 mentions)
Ncl dys3, by Leica (1 mentions)
Gapdh antibody, by Thermo Fisher Scientific (1 mentions)
α actinin antibody, by Merck Group (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
6 protocols using novex nupage gradient gels
1
Western Blot and qPCR Protein and Gene Expression
2
Histone Extraction and Western Blot
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Histones were first acid-extracted as described below. Protein extracts were made in RIPA buffer and quantitated by BCA assay and diluted to equal concentrations and mixed with 4x LDS sample buffer and sample reducing agent (Invitrogen). Polyacrylamide gel electrophoresis was performed on NuPAGE Novex gradient gels (Thermo Fisher) followed by wet transfer to nitrocellulose membranes. Blocking was briefly performed with 5 % non-fat milk and primary antibody was incubated overnight at 4° C in 5 % milk, followed by washing in PBST, then with HRP-conjugated secondary antibody (Cell Signaling) at room temperature for 1 hour followed by washing, then developed with ECL pico or femto kits (Thermo Fisher) and imaged using ChemiDoc Imaging system (Bio-Rad).
3
Dysferlin Quantification in Muscle Samples
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From each muscle, fresh-frozen serial tissue sections were taken for protein preparation. Muscle samples harvested from treated and control groups were compared to WT tissue for levels of vector-mediated dysferlin compared to endogenous dysferlin. Protein (5 μg for WT lanes and 10 μg for all other lanes) extracted from treated and control samples was separated by SDS-PAGE (3–8% Novex NuPAGE gradient gels; Invitrogen), blotted on polyvinylidene fluoride membrane, and probed with NCL-Hamlet (Novocastra) primary antibody (for dysferlin) at a dilution of 1:1,000, or γ-tubulin antibody (Sigma–Aldrich) at a dilution of 1:10,000, followed by HRP-labeled goat anti-mouse IgG (1:5,000; Millipore) or goat anti-rabbit IgG (1:5,000; Millipore), and signal captured on Hyblot CL autoradiography film (Denville). Densitometry analysis was performed using ImageQuant TL.
4
Dystrophin Expression Analysis in mdx Mice
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Western blot analyses were performed as previously reported (Potter et al., 2021 (link)). Protein (50 µg) extracted from LTA and RTA samples from mdx mice were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (3-8% Novex NuPAGE gradient gels, Invitrogen, Waltham, MA, USA), blotted on polyvinylidene fluoride membranes and probed with primary monoclonal antibody specific for dystrophin (NCL-DYS3; Leica Biosystems, Richmond, IL, USA) at a dilution of 1:20 followed by Alexa Fluor 680 goat anti-mouse (1:5000, Licor, Lincoln, NE, USA). For the loading control, a GAPDH antibody (Invitrogen) was used at a dilution of 1:2500 followed by a mouse IgG horseradish peroxidase-linked whole antibody derived from sheep (Millipore Sigma, St. Louis, MO, USA) at a dilution of 1:1000.
5
Dystrophin Western Blot Analysis
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Western blots were performed according to our previously used protocol, with several modifications specific for each antibody used.39 (link) Samples from WT mice, mdx-LR mice, and vector-dosed mdx mice were used for each western blot. Protein (50 μg for muscle and organs) extracted from samples was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (3–8% Novex NuPAGE gradient gels, Invitrogen, Waltham, MA), blotted on polyvinylidene fluoride membrane, and probed with dystrophin primary antibody Dys1 for dystrophin detection and Dys3 for micro-dystrophin (Leica Biosystems) at a dilution of 1:50 and 1:20, respectively, or neuronal nitric oxide synthase (nNOS) primary antibody (Fisher Scientific) at a dilution of 1:1,000. Loading controls used included γ-tubulin antibody (Sigma) or α-actinin antibody (Sigma) at a dilution of 1:10,000 followed by Alexa Fluor 680 goat anti-mouse (1:5,000, LI-COR, Lincoln, Nebraska). Additional detail about antibodies used for western blot analysis is shown in Supplementary Table S1 .
6
Quantitative Dysferlin Protein Analysis
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From each muscle, fresh-frozen serial tissue sections were taken for protein preparation. Muscle samples harvested from treated and control groups were compared with WT tissue for levels of vector-mediated dysferlin compared to endogenous dysferlin. Protein (15 μg) extracted from treated and control samples was separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (3–8% Novex NuPAGE gradient gels, Invitrogen), blotted on PVDF (polyvinylidene fluoride) or nitrocellulose membrane and probed with NCL-Hamlet (Novocastra, Buffalo Grove, IL USA) primary antibody (for dysferlin) at a dilution of 1:1000, or γ-tubulin antibody (Sigma-Aldrich, St. Louis, MO USA) at a dilution of 1:10,000 followed by horseradish-peroxidase (HRP) labeled goat anti-mouse IgG (1:5000; Millipore, Billerica, MA USA) or goat anti-rabbit IgG (1:5000; Millipore) and signal captured on Hyblot CL autoradiography film (Denville, Metuchen, NJ USA). Densitometry analysis was performed using ImageQuant TL (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
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