The collected cells were dissolved in lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, and 0.1 mg/ml proteinase K) for 30 min at room temperature. DNA from the supernatant was extracted by chloroform/phenol/isoamyl alcohol (24/25/1, v/v/v), and was precipitated by ethanol. The extracted DNA was then transferred to 1.5% agarose gel containing 0.1 µg/ml EtBr, and electrophoresis was carried out at 70 V. The gel was visualized using a Chemidox (Bio-Rad Lab., Hercules, CA, USA).
Chemidox
Manufactured by Bio-Rad
Sourced in United States
The ChemiDox is a versatile laboratory equipment designed for chemiluminescence detection. It provides a compact and efficient solution for analyzing various types of samples, including Western blots, ELISA, and other chemiluminescent-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Dmrb microscope, by Leica (1 mentions)
Dc500, by Leica (1 mentions)
Fd8030, by Fudebio (1 mentions)
Polyvinyl fluoride membrane, by Merck Group (1 mentions)
Fdm007, by Fudebio (1 mentions)
Anti s1pr2, by Proteintech (1 mentions)
Anti akt, by Wanlei (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Fdr007, by Fudebio (1 mentions)
Mitochondrial protein extraction kit, by BestBio (1 mentions)
6 protocols using chemidox
1
Genomic DNA Extraction and Electrophoresis
2
Protein Profiling of MSC Fractions
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The protein concentrations of MSC whole-cell lysate, MSC membrane, MSC membrane-formed vesicles, hard MCSNs, and soft MCSNs were measured using BCA assay (BCA Protein Assay Kit, Thermofisher Scientific). Then the samples with the equivalent volume of solution were loaded on Bio-Rad’s Mini-PROTEAN TGX gels, and the protein profiles were examined by Coomassie blue staining. The protein pattern in SDS-PAGE gel was imagined by a ChemiDox (BioRad).
3
Quantifying Caspase-3 and mHTT Inclusions
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Immunostained sections were examined using a Leica DMRB microscope, photographed at a magnification of 10–100 (camera: Leica DC500; Varshaw Scientific, Atlanta, GA, USA) and captured with computer software (SPOT Basic). The percentage of caspase-3 positive neurons was calculated based on number of positive cells per total nuclei. Five randomly captured images at 40× magnification were used to determine the number of caspase-3 positive and total number of nuclei for each monkey. Brain sections were labeled with mEM48 and used to determine the percentage of the number of EM48 (mHTT) positive cells with intranuclear inclusions. Eight randomly captured images at 20× magnification were used to determine the number of total number of EM48 positive cells with intranuclear inclusion for each monkey. For quantitation of protein expression by densitometry with Western blot, bands were captured and the intensity of positive bands was quantified with ChemiDox (BIORAD Inc.). All data are presented as mean ± standard error of the mean (SEM), and analyzed by one-way ANOVA using Graphpad Prism software, and followed by Newman-Keuls method for comparisons between groups.
4
DNA Extraction from EAEG-Treated HeLa Cells
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After treatment of HeLa cells with different concentrations (0, 45, 60, and 75 μg/ml) of EAEG for 24 or 48 h, the phenol/chloroform/isoamyl alcohol was used to extract DNA from cells that had been digested with proteinase K. Cell lysate was collected, incubated at 55°C for 10 h, and then treated with 40 μg/μl DNase-free RNase at 37°C for 40 min. The DNA was precipitated with 4 volumes of ethanol and electrophoresed in 1.5% agarose gels and visualized by ethidium bromide staining using a Chemidox (BioRad, Hercules, CA).
5
Western Blot Analysis of Protein Targets
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Proteins were extracted using RIPA buffer (Beyotime, Shanghai, China), and the concentration was determined by a BCA kit (ThermoFisher Scientific). An equivalent amount of proteins was isolated by SDS-PAGE, and transferred to polyvinyl fluoride membrane (Merck KGaA). After incubation with primary antibodies overnight at 4°C, and incubation with horseradish peroxidase-conjugated secondary antibodies (FDM007 and FDR007, Fudebio, Hangzhou, China) for 2 h. The membranes were treated with the enhanced chemiluminescent reagents (MILLIPORE, WBKLS0500). The signals were examined by ChemiDox (bio-rad, USA) with the treatment of an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). The primary antibodies involved in the present study were GAPDH (1:1000, Abcam), anti-S1PR2 (1:500, Proteintech), anti-AKT (1:1000, Wanleibio), anti-p-AKT (Ser473) (1:1000, Wanleibio).
6
Gastrocnemius Muscle Protein Analysis
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WB analysis of gastrocnemius muscles was performed as previously reported [21 (link)]. If mitochondrial protein isolation is required, it is done using the mitochondrial protein extraction kit (BB-3171, Best Bio, Shanghai, China). Primary antibodies include Stat3 (1 : 1000), P-Stat3 (1 : 1000), HIF-1α (1 : 1000), BNIP3 (1 : 5000), Beclin 1 (1 : 1000), Atg7 (1 : 1000), LC3A/B (1 : 1000), p62 (1 : 1000), GAPDH (1 : 2500), and COX IV (1 : 2000). After the primary antibody protocol was completed, the corresponding secondary antibody (1 : 5000) was added according to the protocol. The density values of the bands were captured and documented through a gel image analysis system (ChemiDox™, Bio-Rad, USA) and normalized to GAPDH or COX IV; n = 3.
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