A total of 25 μg of each venom sample was evaluated by polyacrylamide gel electrophoresis (12.5%) in the presence of SDS, under reducing and non-reducing conditions (Laemmli, 1970 (link)), in a Mini-Protean II system (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, venom samples were reconstituted in 0.063 M Tris-HCl buffer, pH 6.8, containing 2% SDS, 5% glycerol and 0.001% bromophenol blue and then boiled for 4 min before electrophoresis at 100 V (constant). After electrophoresis, the gels were stained with 0.1% brilliant blue Coomassie R250 in acetic acid/ethanol/water (5:25:70, v/v) and then distained in the same solution. The analysis was performed using the BioRad Quantity One 1-D Analysis Software Version 4.6.7 program.
Quantity one 1 d analysis software version 4
Manufactured by Bio-Rad
Sourced in United States
Quantity One 1-D Analysis Software, version 4.4 is a software tool designed for the analysis of one-dimensional electrophoresis and blot data. It provides basic functions for image acquisition, data analysis, and visualization of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc eq, by Bio-Rad (2 mentions)
Mini protean 2 system, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Srebp1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Horseradish peroxidase coupled anti rabbit igg, by GE Healthcare (1 mentions)
Ne per nuclear protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Endothelial cell growth medium, by Provitro (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
X ray film, by Kodak (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bicinchoninic acid kit, by Beyotime (1 mentions)
X ray film, by Fujifilm (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Chemiluminescent horseradish peroxidase substrate, by Thermo Fisher Scientific (1 mentions)
13 protocols using quantity one 1 d analysis software version 4
1
Venom Protein Characterization by SDS-PAGE
2
Analyzing Venom Profiles of B. venezuelensis
3
Western Blot for PMCA, GAP43, Calmodulin, and Calcineurin
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For Western blot, 40–60 μg or 80 μg (for phosphoprotein detection) of protein lysate were resolved on a 8 % SDS-PAGE and transferred onto nitrocellulose membrane using a semi-dry method. Membranes were first blocked with 3 % bovine serum albumin (BSA) in TBS-T (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.05 % Tween-20) for 1 h at room temperature and then overnight incubated with primary antibodies at 4 °C. The following primary antibodies were used: polyclonal anti-PMCA1, anti-PMCA2, anti-PMCA3 (diluted 1:1000), monoclonal anti-PMCA4 (1:1000), polyclonal anti-GAP43 (1:1000), polyclonal anti-phosphoGAP43 (1:750), monoclonal anti-calmodulin (1:1500), and polyclonal anti-calcineurin A (1:1000). Polyclonal anti-GAPDH (1:1000) antibodies were used to standardize each line and as an integral loading control. Following 3 × 15 min washes with TBS-T, membranes were incubated for 2 h at room temperature with species-specific secondary antibodies (1:5000) conjugated with alkaline phosphatase. Bands were visualized with BCIP/NBT used according to the manufacturer’s protocol. Blots were densitometrically quantified using GelDocEQ with Quantity One 1-D Analysis Software version 4.4.1 (Bio-Rad).
4
Western Blot Analysis of Protein Expression
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Scraped cells were resuspended in RIPA buffer supplemented with 1 mM PMSF, 2 mM Na3VO4 and protease inhibitor cocktail and lysed for 30 min on ice. Then, lysates were centrifuged at 10 000× g for 5 min and supernatants were boiled for 5 min in Laemmli buffer. Total protein content was quantified using Bio-Rad Protein Assay.
20 µg of total cellular proteins were resolved on a 10% SDS-PAGE and transferred onto nitrocellulose membrane using semi-dry method. Membranes were first blocked with 6% BSA in TBS-T buffer (10 mM Tris-HCl, pH. 7.4, 150 mM NaCl, 0.05% Twwen-20) for 1 h at room temperature and then probed overnight at 4°C with primary antibodies against GFP (1∶1000), PMCA2 (1∶750), PMCA3 (1∶750) and GAPDH (1∶1000). Following several washes with TBS-T, membranes were incubated with appropriate secondary antibodies (1∶5000) coupled with alkaline phosphatase at room temperature for 4 h. Bands were visualized using Sigma Fast BCIP/NBT used according to the manufacturer's instructions. Blots were scanned and the bands intensity was measured using GelDocEQ with Quantity One 1-D Analysis Software version 4.4.1 (Bio-Rad).
20 µg of total cellular proteins were resolved on a 10% SDS-PAGE and transferred onto nitrocellulose membrane using semi-dry method. Membranes were first blocked with 6% BSA in TBS-T buffer (10 mM Tris-HCl, pH. 7.4, 150 mM NaCl, 0.05% Twwen-20) for 1 h at room temperature and then probed overnight at 4°C with primary antibodies against GFP (1∶1000), PMCA2 (1∶750), PMCA3 (1∶750) and GAPDH (1∶1000). Following several washes with TBS-T, membranes were incubated with appropriate secondary antibodies (1∶5000) coupled with alkaline phosphatase at room temperature for 4 h. Bands were visualized using Sigma Fast BCIP/NBT used according to the manufacturer's instructions. Blots were scanned and the bands intensity was measured using GelDocEQ with Quantity One 1-D Analysis Software version 4.4.1 (Bio-Rad).
5
Protein Extraction and Western Blotting
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Cells were collected by centrifugation, washed with PBS and total protein extracts were obtained by lysing them for 20 min at 4°C in a buffer consisting of 20 mM Tris-HCl (pH 7.5) containing 137 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, and 1% Nonidet P-40, and supplemented with a protease inhibitor cocktail, 1 mM sodium orthovanadate, and 10 mM NaF. After brief sonication and centrifugation for 15 min at 10,000×g at 4°C, the supernatants were collected, and samples containing equal amounts of proteins were resolved by SDS-polyacrylamide gel electrophoresis. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes and immunodetected, as previously described [30] (link). When convenient, the relative band intensities were quantified using the Quantity One 1-D Analysis Software, version 4.6 (Bio-Rad Laboratories, Inc, Hercules, CA).
6
Protein Extraction and SDS-PAGE Immunoblotting
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Cells were collected by centrifugation, washed with PBS and total protein extracts were obtained by lysing them for 20 min at 4 °C in a buffer consisting of 20 mM Tris–HCl (pH 7.5) containing 137 mM NaCl, 2 mM EDTA, 10 % (v/v) glycerol, and 1 % Nonidet P-40, and supplemented with a protease inhibitor cocktail, 1 mM sodium orthovanadate, and 10 mM NaF. After brief sonication and centrifugation for 15 min at 10,000×g at 4 °C, the supernatants were collected, and samples containing equal amounts of proteins were resolved by SDS–polyacrylamide gel electrophoresis. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes and immunodetected, as previously described [28 (link)]. When convenient, the relative band intensities were quantified using the Quantity One 1-D Analysis Software, version 4.6 (Bio-Rad Laboratories, Inc., Hercules, CA).
7
Quantifying Liver Protein Regulation
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Total liver extracts were prepared as for MTHFR assays (see previous paragraph). Nuclear and cytoplasmic fractions were isolated by using the NE-PER Nuclear Protein Extraction Kit (Thermo Scientific) following the manufacturer’s instructions. Protein concentration was determined by Bradford assay by using bovine serum albumin as a standard. Western blotting was performed with conventional methods by using 25 μg total or cytoplasmic protein or 15 μg nuclear protein. Primary antibodies were sterol regulatory element-binding protein (SREBP-1; Santa Cruz Biotechnology), cyclic AMP-responsive element-binding protein (Cell Signaling Technology), β-actin (Sigma-Aldrich), and MTHFR (35 (link)). Secondary antibody was horseradish peroxidase–coupled anti–rabbit IgG (GE Healthcare). Bands were visualized with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) and film exposure and quantified by densitometry by using Quantity One 1-D Analysis Software version 4.6.9 (Bio-Rad). Results were normalized to the β-actin loading control and reported relative to the mean value for CD-fed Mthfr+/+ mice, which was standardized to a reference value of 1.
8
NF-κB and ICAM-1 Protein Expression
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Myocardial tissue (100 mg) was lysed using radio-immunoprecipitation assay buffer to extract proteins. Protein concentration was determined by the Bradford method. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and were transferred to a polyvinylidene fluoride membrane. After blocking with skim milk, the membrane was incubated with NF-κB and ICAM-1 monoclonal antibodies overnight, followed by incubation with secondary antibodies for 1 h. The membrane was developed by using chemiluminescence (Amersham, Piscataway, NJ, USA) and was exposed in a dark room. The Quantity One 1-D analysis software version 4.6.9 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for the data analysis. The results are presented as relative expression level: the ratio of protein gray value to β-actin gray value.
9
Integron Amplification and Restriction Digestion
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Amplified product of variable region of class 1 integron (1.6 kb) from Pseudomonas spp. DF5TC, Cronobacter spp. DF52TC, Alcaligenes spp. DF19TB, Stenotrophomonas spp. DF3SA, K. pneumoniae DF12SA, E. coli DF30TA, Providencia spp. DF1SB, Alcaligenes spp. DF43SB, S. flexneri DF1TA, S. aureus DF8TA, Enterococcus spp. DF5SB, and Enterococcus spp. DF16SA was purified and digested with AluI and RsaI following the instructions of the manufacturer (New England BioLabs). Restriction digestion was done in a final volume of 25 μl containing 1 x restriction enzyme buffer, 0.30 μl (3.0 U) restriction enzyme and 15 μl PCR product. After mixing, samples were incubated for 6 h in a water bath preset at 37°C. Reaction was terminated by heat inactivation of restriction enzymes at 70°C for 20 min. The digested products were run in a 3% agarose gel at 100 V in TAE buffer for 4–5 h. Cluster analysis of restriction fragment length polymorphism (RFLP) types was performed by the unweighted pair-group method with arithmetic means using Quantity One 1-D Analysis Software, version 4.4 (BioRad).
10
Endothelial Cell Response to Microparticles
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HUVECs were stimulated for 6 h with microparticles from resuscitated or healthy subjects, or with the cell free supernatant of resuscitated patients, respectively, in endothelial cell growth medium (Provitro) with 10% fetal bovine serum (FBS). Cell lysates were resolved on a reducing polyacrylamide gel, plotted onto a nitrocellulose membrane (Amersham GE Healthcare, Pittsburgh, PA, USA) and blocked with 3% non-fat milk in PBS/Tris with 0.1% Tween 20 for 2 h at room temperature (20 to 22°C). The membrane was then incubated with primary antibody overnight at 4°C. After 1 h of incubation with the secondary antibody, proteins were visualized using ECL reagent (Amersham GE Healthcare). Densitometric analysis of western blots was performed using Quantity One 1-D Analysis Software Version 4.4 (Bio-Rad Laboratories GmbH, Munich, Germany). All western blots were repeated at least three times and quantified data are shown.
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