Heart tissue lysates were homogenized using a polytron tissue disruptor in cell lysis buffer (Cell Signaling Technologies cat. #9803) with protease cocktail inhibitor (Thermo Sci. cat. #87786). Proteins were extracted from H9C2 cardiomyoblasts and heart tissue with cell lysis buffer and supplemented with protease cocktail inhibitor. Proteins were standardized using a Nanodrop (Thermo Scientific). Changes in FXN levels were measured by western analysis as described previously using FXN (1:1,000, SantaCruz, cat. #25830) antibody (Mouli et al., 2015 (link); Nanayakkara et al., 2015 (link)). Briefly, protein lysates were denatured, resolved and blotted on a nitrocellulose membrane. The blots were stained with primary antibodies, FXN (1:1,000, Santa Cruz, cat. #25830) and α-tubulin (1:2,000, DSHBY, cat. #12G10), washed with Tris-buffered saline-Tween and incubated with a secondary - horseradish peroxidase-conjugated antibody (1:2,000, Rockland) for 1 h. Blots were visualized with chemiluminescence reagent (Millipore) and imaged on a Bio-Rad gel dock system. Protein bands were analyzed using Quantity One software (BioRad) or ImageJ software and standardized to α-tubulin.
Gel dock system
Manufactured by Bio-Rad
The Gel Dock System is a laboratory equipment used for capturing and analyzing images of electrophoresis gels. It provides a platform for visualizing and documenting the results of gel-based experiments, such as DNA, protein, or RNA analysis. The core function of the Gel Dock System is to enable researchers to capture high-quality digital images of their gel samples, which can then be further analyzed using specialized software.
Automatically generated - may contain errors
Lab products found in correlation
Polyacrylamide gel, by Bio-Rad (2 mentions)
Reagent based technique, by Thermo Fisher Scientific (2 mentions)
Anti ube3a, by Cell Signaling Technology (2 mentions)
Anti α tubulin, by Cell Signaling Technology (2 mentions)
Anti tata binding protein tbp, by Cell Signaling Technology (2 mentions)
α tubulin, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence reagent, by Merck Group (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Imagej software, by Bio-Rad (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
5 protocols using gel dock system
1
Quantifying Frataxin Levels in Heart Tissues
2
Protein Detection via Western Blot
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Proteins were separated by SDS–polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Immobilon-P, EMD Millipore) by liquid transfer, and the Western blots were probed using the appropriate primary antibodies (1:1000) followed by alkaline phosphatase secondary antibodies (1:5000). The signals were detected using a chemiluminescence system (Thermo Fisher Scientific), followed by Gel Dock system (Bio-Rad) imaging.
3
Subcellular Fractionation and Ube3a Quantification
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Nuclear and cytosolic fractions of cortical samples (Extended Data Fig. 2d ) were separated using a commercially available reagent-based technique (Fisher Scientific) according to manufacturer instructions. Proteins were loaded onto 4–15% polyacrylamide gels (Bio-Rad), transferred to PVDF membranes, blocked with 5% nonfat milk in TBS-T, and incubated overnight at 4 degrees with the following antibodies; anti-Ube3a (#7526, Cell Signaling), anti-alpha-Tubulin (#3873, Cell Signaling), and anti-TATA-binding protein (TBP; #8515, Cell Signaling). After TBS-T washes, blots were incubated with species-specific HRP-conjugated secondary antibodies for 1–2 hours at room temperature. Blots were developed with Pico chemiluminescent reagent (Pierce) and digital images were acquired using the gel dock system (Bio-Rad). Following subcellular fractionation, nuclear and cytosolic Ube3a levels were normalized to levels of TBP and alpha-Tubulin, respectively.
4
Expression and Purification of MtbFtsZ Protein
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pSAR 1 plasmid harboring MtbFtsZ gene in pET15 vector is a kind gift from Dr. Malini Rajagoplan, University of Texas, USA. Protein was expressed in E. coli BL21 (DE3) Rosetta cells. MtbFtsZ is a soluble protein and it was expressed by induction of IPTG, and purified by Ni-NTA column chromatography (Chen et al., 2007 (link); Duggirala et al., 2016 (link)). The protein was analyzed on SDS-PAGE and its purity was determined to be >95%. The gel was visualized in BioRad gel dock system and Quantity one 4.6.9 software. Protein concentration was measured by BCA method and stored at −80°C until further use The protein was stable in 25 mM HEPES buffer (pH 6.5), 50 mM KCl and 5 mM MgCl2. (Rajagopalan et al., 2005 (link)).
5
Subcellular Fractionation and Ube3a Quantification
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