Cells were collected and lysed on ice using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing 1% PMSF (Beyotime Institute of Biotechnology). The protein concentration was determined using the bicinchoninic acid method. In total, 50 µg of total protein was separated using 10% SDS-PAGE and transferred onto PVDF membranes. After 5% bovine serum albumin (cat. no. ST023; Beyotime Institute of Biotechnology) blocking at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies. The primary antibodies used were: anti-ST3Gal3 (1:500 dilution; cat. no. SC-134040; Santa Cruz Biotechnology, Inc.), anti-caspase-8 (1:1,000 dilution; cat. no. 4790; Cell Signaling Technologies, Inc.), anti-caspase-3 (1:1,000 dilution; cat. no. 14220; Cell Signaling Technologies, Inc.) and anti-GAPDH (1:2,000 dilution; cat. no. AF0006; Beyotime Institute of Biotechnology). Membranes were then incubated with secondary anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3,000 dilution; cat. nos. A0208 and A0216; Beyotime Institute of Biotechnology) for 1 h at room temperature. Results were acquired using the Gel Logic 1500 imaging system (Kodak). GAPDH was used as a loading control.
Gel logic 1500 imaging system
Manufactured by Kodak
Sourced in United States, United Kingdom, Italy
The Gel Logic 1500 Imaging System is a laboratory equipment designed for imaging and analyzing gel electrophoresis samples. It provides high-resolution imaging capabilities for a variety of gel types, including agarose and polyacrylamide gels. The system's core function is to capture, digitize, and analyze gel images with precision and consistency.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (7 mentions)
Polyvinylidene difluoride membrane, by PerkinElmer (4 mentions)
Liteablot extend substrate, by Euroclone (3 mentions)
Nupage gel, by Thermo Fisher Scientific (3 mentions)
Mouse and rabbit horseradish peroxidase conjugated igg, by Bio-Rad (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Ecl western blotting kit, by Cytiva (2 mentions)
Horseradish peroxidase labeled secondary antibody, by Bio-Rad (2 mentions)
Transignal protein dna assay kits, by Panomics (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Anti caspase 8, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Genescreen plus, by PerkinElmer (1 mentions)
Hybond n nylon membrane, by Cytiva (1 mentions)
Phusion hot start 2 high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (1 mentions)
Sds polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
23 protocols using gel logic 1500 imaging system
1
Western Blot Analysis of Apoptosis Markers
2
BAC Clones Southern Blot Analysis
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BAC clones were digested with SalI and NotI, and fragments were separated by gel electrophoresis and transferred to a GeneScreen Plus hybridization membrane (Perkin-Elmer, Waltham, MA, USA) by capillary blotting [35 (link),54 ]. The filter was evaluated with 32P-riboprobes generated with RNA polymerases from linearized gene clones that served as templates to incorporate 32P labeled ribonucleotides as described in [23 (link),35 (link),55 (link)]. The gene clones chosen for templates had an A6 element pattern (GenBank accession number EF607716.1), a B3 element pattern (EF607770.1), and a D1 element pattern (EF607784.1) [19 (link)]. After hybridization with the probes, the filter was exposed to X-ray film at −80 °C, which was processed with developer and fixer, scanned with epi-white light, and imaged with the Kodak Gel Logic 1500 Imaging System.
3
Genotyping AAVS1 Locus Modification
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Junction PCRs were performed using locus-specific genomic primers that bind outside of the AAVS1 homology regions, in combination with donor vector-specific primers (Supplementary Table S1 ). The fragments were amplified by Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific). For Southern blot analysis, 5 μg genomic DNA was digested overnight with ApaI or NcoI restriction enzyme (NEB) and separated on agarose gel, then transferred onto Hybond N + nylon membrane (Amersham). DIG-labeled DNA probes were prepared by random primed labeling for the AAVS1 left and right homology regions (581 and 622 bp, respectively). Probe labeling, hybridization and detection were performed using DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche), following instructions of the manufacturer. Kodak Gel Logic 1500 Imaging System was used to document the gels and blots.
4
Protein Extraction and Immunoblotting Protocol
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Tissues and cells were homogenized in lysis buffer (20 mM Tris-Cl, 5 mM EGTA, pH 7.5 and protease inhibitor cocktail all from Sigma-Aldrich) and the protein content of samples was measured by the BCA protein assay kit (Pierce, Rockford, IL). The lysates containing 20 μg total protein (except for RasGRP3 and phosphoRasGRP3 immunoblotting where 60 μg total protein was used) were separated by electrophoresis on 10% SDS-polyacrylamide gels (Invitrogen) and transferred onto BioBond nitrocellulose membranes (Whatman, Maidstone, UK). After the membranes were blocked and labeled with the appropriate primary and secondary antibodies, the immunoreactive bands were visualized by SuperSignal West Femto Chemiluminescent Substrate-enhanced chemiluminescence (Pierce, Rockford, IL) using a Gel Logic 1500 Imaging System (Kodak, Tokyo, Japan). To assess equal loading (and to obtain an endogenous control), membranes were re-probed with a GAPDH or actin β antibody followed by a similar visualization procedure as described above.
5
Protective Properties of LNPs Against pDNA Degradation
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To assess the protective properties of LNPs against enzymatic degradation of pDNA we performed the DNase I Protection Assay using a DNase I, RNase-free enzyme (Thermo Scientific, EN0521). LNP-encapsulated pDNA or naked pDNA at an amount of 1 µg were incubated with DNase I for 60 minutes at 37 °C. Naked pDNAs were used as positive control. The reaction volume was made up to 10 µL using ultrapure sterile water. Then, 5 µL EDTA was added and incubated for 60 minutes at 65 °C to stop the reaction. Following, isopropyl alcohol was added at 5:1 proportion (v/v) alcohol:LNP, when applied. Next, 2.5 µL (50 UI) of heparin was added in each sample and incubated for 60 minutes at room temperature. All samples were run by gel electrophoresis on 0.8% agarose stained with SYBR Safe in ×0.5 Tris-acetate-EDTA (TAE) buffer for 140 minutes. The gel was visualized and documented using Kodak Gel Logic 1500 Imaging System.
6
Protein Expression Profiling by Immunoblot
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Total MMEC protein lysates were quantified with the Bradford assay (Bio-Rad, Hercules, CA, USA) and subjected to immunoblot with primary and secondary antibodies to the following: cMet (cod.#8198), phospho (p)-cMet (cod.#3077), VEGFR2 (cod.#9698), p-VEGFR2 (cod.#3770), p-extracellular signal regulated kinase (p-Erk)-1/2 (cod.#4370), Erk-1/2 (cod.#9102), p-Protein kinase B (p-AKT) (cod.#4060), AKT (cod.#9272), p-Phosphoinositide-specific phospholipase C (p-PLC)γ1 (cod.#8713), PLCγ1(cod.#5690), p-P38 mitogen-activated protein kinases (p-p38) (cod.#4511), p38 (cod. #8690) (Cell Signaling Technology, Danvers, MA, USA); beta-actin (Sigma-Aldrich) (cod. A1978); and mouse and rabbit horseradish peroxidase–conjugated IgG (Bio-Rad). Immunoreactive bands were visualized by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific, Waltham, MA, USA) and the Gel Logic 1500 Imaging System (Eastman Kodak Co.), quantified with the Kodak Molecular Imaging Software, and expressed as arbitrary optical density (OD).
7
Western Blot Protein Quantification and Detection
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Cellular lysates were obtained solubilising about 3 × 10 6 of cells in 0.1 ml of lysis buffer (1% Triton-X-100, 0.5 mM EDTA, 0.6 mM PMSF, 100 μl/ml of protease inhibitor cocktail dissolved in phosphate buffered saline at pH 7.4), sonicated and centrifuged at 10000 ×g. The protein content of the supernatant (cell lysate) was determined according to Bradford (Bradford 1976 ) and 60 μg of proteins were separated by 12% SDS-PAGE. Gels were transferred onto nitrocellulose membrane (BioRad) for 1 h at 100 V. The nitrocellulose membranes were incubated overnight at 4 °C with the indicated antibody. Primary antibodies were visualized using horseradish peroxidase-conjugated secondary antibodies (1:2000) . The chemiluminescence signals were revealed using an ECL Western blotting kit (Amersham Bioscience, Buckinghamshire, UK) and measured with Gel Logic 1500 Imaging System, Kodak.
8
Immunoblotting of Transmembrane Proteins
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Protein lysates from MGECs and MMECs were obtained with a lysis buffer that preserves transmembrane proteins. Thirty-five micrograms of protein lysates was separated on 4%-12% NuPAGE gels (Invitrogen Corp.), electrotransferred to a polyvinylidene difluoride membrane (PerkinElmer Life Science Inc., Boston, MA), immunoblotted overnight with primary antibodies (Supplementary Table 1), and incubated with horseradish peroxidase–labeled secondary antibodies for 1 hour (Bio-Rad, Hercules, CA). Immunoreactive bands were visualized by enhanced chemiluminescence (Bio-Rad) with the Gel Logic 1,500 Imaging System (Eastman Kodak Co., Rochester, NY), and quantified as optical density units with Kodak Molecular Imaging Software.
9
PPAR β/δ Protein Quantification
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Following MGEC and MMEC lysis using RIPA lysis and extraction buffer (Thermo Fisher Scientific), total protein extracts (30 µg) were subjected to SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Immunoblots were performed overnight using the following antibodies: anti-PPAR β/δ antibody (Thermo Fisher Scientific), anti-β-actin antibody (Sigma-Aldrich), and mouse and rabbit horseradish peroxidase-conjugated anti-IgG (Bio-Rad). The immunoreactive bands were detected by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) using the Gel Logic 1500 imaging system (Eastman Kodak, New York, NY, USA). Densitometry analysis of band intensity was performed using Kodak molecular imaging software 5.0 (Kodak, New York, NY, USA). The results are reported as the relative density.
10
Immunoprecipitation and Western Blot Analysis of ec-Mpl Protein
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Total protein lysate (600 mg) from BMECs was immunoprecipitated using antiec-Mpl polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX) with Immunoprecipitation Dynabeads Protein G Kit (Invitrogen Corp., Carlsbad, CA), according to the manufacturer's instructions. Next, samples were analyzed by Western blot analysis: they were separated on 4% to 12% NuPAGE gels (Invitrogen Corp.), electrotransferred to a polyvinylidene difluoride membrane (Per-kinElmer Life Science Inc., Boston, MA), and immunoblotted with antiec-Mpl monoclonal antibody (Santa Cruz Biotechnology). b-Actin was used as loading control, and analyzed on total lysate using antieb-actin antibody (Sigma-Aldrich). Then, the membranes were incubated with horseradish peroxidaseelabeled secondary antibodies (Bio-Rad, Hercules, CA). Immunoreactive bands were visualized by enhanced chemiluminescence (LiteAblot extend substrate; Euroclone, Pero, Italy) and the Gel Logic 1500 Imaging System (Eastman Kodak Co, Rochester, NY).
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