Cells were washed with 1xPBS and lysed in protein sample buffer (100 mM Tris-HCl pH = 6.8, 4% SDS, 20% glycerol, 0.2 M DTT), boiled 5 min at 95 °C and vortexed to shear genomic DNA. Proteins were separated using SDS-PAGE and subsequently transferred onto a nitrocellulose membrane (GE Healthcare) by tank transfer. Membranes were blocked using 5% BSA, primary antibodies incubated overnight at 4 °C and secondary antibodies for 3 h at 4 °C. All western blots were developed with freshly mixed ECL solutions (GE Healthcare). Antibodies used in this study are the following: Phospho-p70 S6 Kinase (Thr389) from Cell Signaling (#9234), 4E-BP1 from Cell Signaling (#9452), Phospho-4E-BP1 (Thr37/46) from Cell Signaling (#2855), Tubulin from Sigma Aldrich (T6199). Western blot bands were quantified using ImageJ.
Ecl solution
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden, Germany
ECL solution is a chemiluminescent detection reagent commonly used in Western blot analysis to visualize and quantify proteins. It contains a luminol-based substrate that reacts with horseradish peroxidase (HRP) enzyme to produce light, allowing the detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (7 mentions)
Protein assay method, by Bio-Rad (5 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Skim milk, by Bio-Rad (4 mentions)
Mini protein tgx gel, by Bio-Rad (4 mentions)
Anti β actin, by Merck Group (3 mentions)
Chemidoc xr, by Bio-Rad (3 mentions)
Amersham imager 600, by GE Healthcare (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti cd63, by System Biosciences (3 mentions)
Imagequant las 500, by GE Healthcare (2 mentions)
0.45 μm nc membrane, by Merck Group (2 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Merck Group (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Skim milk, by Merck Group (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
Maid 2 clone, by Thermo Fisher Scientific (2 mentions)
Za001, by Thermo Fisher Scientific (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
74 protocols using ecl solution
1
Western Blot Analysis of mTOR Pathway
2
SDS-PAGE and Western Blot Analysis
3
Western Blot Detection of Endogenous Proteins
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Proteins were detected by Western blotting according to standard protocols. For detection of the endogenous ZC3H11 protein only cytoskeleton-free extracts were used. Antibodies used were to the ZC3H11 (rabbit, 1:10000, [13 (link)]), RBP6 [19 (link)] and PTP1 [20 (link)]. Detection was done using ECL solutions (GE Healthcare).
4
Trypanosome Protein Interaction Assays
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Sub-cellular fractionation (46 ), dephosphorylation assays (47 (link)), tandem affinity purification (TAP) (43 (link),48 (link)) and co-immunoprecipitation assays (49 (link)) were done as previously described. Phosphatase inhibitors used were Sodium Orthovanadate (2 mM) and Sodium Fluoride (8 mM). For co-immunoprecipitation with V5-ZC3H11, 4 × 107 procyclic trypanosomes or 5 × 107 bloodstream forms were pre-treated for 1 h with the proteasome inhibitor MG-132 (Calbiochem) at a concentration of 10 µg/ml. Cells were lysed in hypotonic buffer (10 mM NaCl, 10 mM Tris-Cl pH7.5, 0.1%NP40 with complete protease inhibitor (Roche), and precipitation was with anti-myc (Biomol) or anti-V5 (Biomol) -coupled beads after the salt was adjusted to 150 mM NaCl.
Proteins were detected by western blotting. Antibodies used were to the V5 tag (AbD seroTec, 1:1000), the Myc tag (Santa Cruz Laboratories, 1:1000), aldolase (rabbit, 1:50000 (50 (link))) ribosomal protein S9 (rabbit antibody raised against recombinant protein prepared by C. Helbig, ZMBH) and trypanothione reductase (kind gift of Prof. L Krauth-Siegel, BZH, Heidelberg). Detection was done using ECL solutions (GE Healthcare). CAT was measured in a kinetic assay involving partition of 14C-buturyl chloramphenicol from the aqueous to the organic phase of scintillation fluid (51 (link)).
Proteins were detected by western blotting. Antibodies used were to the V5 tag (AbD seroTec, 1:1000), the Myc tag (Santa Cruz Laboratories, 1:1000), aldolase (rabbit, 1:50000 (50 (link))) ribosomal protein S9 (rabbit antibody raised against recombinant protein prepared by C. Helbig, ZMBH) and trypanothione reductase (kind gift of Prof. L Krauth-Siegel, BZH, Heidelberg). Detection was done using ECL solutions (GE Healthcare). CAT was measured in a kinetic assay involving partition of 14C-buturyl chloramphenicol from the aqueous to the organic phase of scintillation fluid (51 (link)).
5
Bisulfite Sequencing and Western Blot Analysis
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Bisulfite conversion was performed using EZ DNA methylation kit (Zymo research) according to the manufacturer's instructions. PCR was performed using HotStarTaq DNA Polymerase (QIAGEN), and resulting PCR products were cloned into pCRII TOPO vector (Invitrogen) or pGEM-T (Promega). Individual clones were subjected to sequencing. Sequencing data were analyzed using QUMA (http://quma.cdb.riken.jp). CpG sites within LTR promoter of pMXs we used for bisulfite sequencing have been previously reported (Fussner et al., 2011; Kim et al., 2020) . Primers are listed in Table S1. monoclonal anti-DNMT3B (1:5,000, IMG-184A; NOVUS) and mouse monoclonal anti-TUBULIN (1:5,000, T6199; Sigma-Aldrich). The membranes were then washed three times with 0.1% Tween-20/PBS (PBST) and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies, donkey polyclonal anti-rabbit IgG HRP (NA934; GE Healthcare) and goat polyclonal antimouse IgG+IgM (H+L) HRP (115-035-044; Dianova), for 1 hr at room temperature. The membranes were washed three times with PBST, incubated with ECL solutions (GE Healthcare) and exposed to X-ray films (GE Healthcare).
6
Western Blot Analysis of Ovarian and Embryonic Proteins
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Ovaries from 30 females were collected and homogenized in lysis buffer (20mM Hepes pH7.9, 100mM KCl, 0.1mM EDTA, 0.1mM EGTA, 5% Glycerol, 0.05% Igepal and protease inhibitors (Roche)). The protein extracts were cleared by centrifugation and stored at -80°C.
Eggs were collected every 30 min, dechorionated in bleach and quickly frozen in liquid nitrogen. Protein extracts were prepared from ca. 10 μl of embryos. Protein samples were run on 15% SDS polyacrylamide gel and transferred to Immun-Blot® PVDF membrane (Bio-Rad) for 1h at 60V. Membranes were blocked for 1h at room temperature in 5% non-fat milk in PBS 1X-Tween20 0.05%, followed by an overnight incubation with the primary antibody at 4°C in 5% non-fat milk in PBS1X-Tween20 0.05%. Secondary antibodies used were added and incubated for 2 hours at room temperature. Protein detection was performed using ECL solution according manufacturer’s instruction (GE Healthcare). Antibodies used were: rabbit polyclonal anti-DHD (1/1000) [6 (link)], mouse monoclonal anti-α-Tubulin (Sigma Aldrich #T9026, 1:500), HRP-conjugated goat anti-mouse (Biorad #170–5047; 1:50 000) and peroxidase-conjugated goat anti-rabbit (Thermoscientific #32460; 1:20 000).
Eggs were collected every 30 min, dechorionated in bleach and quickly frozen in liquid nitrogen. Protein extracts were prepared from ca. 10 μl of embryos. Protein samples were run on 15% SDS polyacrylamide gel and transferred to Immun-Blot® PVDF membrane (Bio-Rad) for 1h at 60V. Membranes were blocked for 1h at room temperature in 5% non-fat milk in PBS 1X-Tween20 0.05%, followed by an overnight incubation with the primary antibody at 4°C in 5% non-fat milk in PBS1X-Tween20 0.05%. Secondary antibodies used were added and incubated for 2 hours at room temperature. Protein detection was performed using ECL solution according manufacturer’s instruction (GE Healthcare). Antibodies used were: rabbit polyclonal anti-DHD (1/1000) [6 (link)], mouse monoclonal anti-α-Tubulin (Sigma Aldrich #T9026, 1:500), HRP-conjugated goat anti-mouse (Biorad #170–5047; 1:50 000) and peroxidase-conjugated goat anti-rabbit (Thermoscientific #32460; 1:20 000).
7
Quantifying Monocyte Chemotactic Protein-1 in Ischemic Brain
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Protein lysates were obtained from dissection of ischemic (ipsilateral hemisphere) and healthy (contralateral hemisphere) tissues and subjected to SDS-PAGE prior to western blot analysis. Anti-mouse monocyte chemotactic protein-1 (MCP-1) (1:1000; Cell Signaling) and anti-mouse β-actin (1:40000; EMD Millipore) primary antibodies were used prior to detection by horseradish peroxidase (HRP)-conjugated secondary antibodies and revelation by enhanced chemiluminescence plus (ECL) solution (GE Healthcare Life Sciences). Blots were digitized and proteins level were densitometrically analyzed with ImageJ software.
8
Evaluating Subfraction 5 Cytotoxicity on HepG2 Cells
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HepG2 cells were seeded into a 102 cm dish at a density of 1 × 106 cells/dish and incubated with various concentrations of subfraction 5 (0, 50, 100, 200, and 400 μg/mL) and 5-FU (25 μg/mL) for 24 h. The cells were harvested by cell scraping, rinsed with ice-cold PBS twice, and centrifuged at 3,000 rpm for 5 min. Pellet cells were lysed in a cold modified RIPA buffer with a protease inhibitor cocktail and then vortexed for 20 min at 4 °C. After centrifugation at 12,000 rpm for 20 min at 4 °C, the supernatant was collected to measure the total protein concentration using the Coomassie Plus (Bradford) Assay Kit (Thermo Science, Rockford, IL, USA). The protein samples were isolated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. Next, they were blocked with 5% bovine serum protein (BSA) in TRIS-buffered saline with Tween 20 (TBST) for 1 h and soaked in primary antibodies (1 : 1000 dilutions) overnight in a 4 °C microplate shaker. The membranes were washed with TBST three times subsequently dipped in secondary antibodies for 1 h, and washed again with TBST three times. Finally, they were visualized with ECL solution (GE Health Care, Buckinghamshire, UK), and the protein levels were analyzed using Image J software (National Institutes of Health, USA).
9
Protein Extraction and Western Blot Analysis
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Total protein extraction and electrophoresis were performed as before [21 (link)]. The primary antibody reaction was done overnight at 4 °C with the following primary antibodies (excepting ALDH1A3 antibody, all from Cell Signaling Technology): anti-ALDH1A3 (1:1000, Novus Biologicals), anti-STAT3 (1:1000), anti-phospho-STAT3 (Tyr705) (p-STAT3) (1:2000), anti-GAPDH (1:1000), anti-phospho-Akt (Ser437) (p-Akt) (1:1000), anti-PTEN (1:1000). After the second antibody reaction, the blots were incubated with ECL solution (GE Healthcare) and the image was acquired by using ImageQuant LAS 500 (GE Healthcare, Freiburg).
10
Albiflorin and PL Impact on MC3T3-E1 Cells
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MC3T3-E1 cells were cultured in 60π dishes (5 × 105 cells) and incubated in osteogenic medium with or without albiflorin and PL for 2 days. The cells were washed 3 times using DPBS and then dissolved in RIPA buffer (50 mM Tris-Cl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS with protease inhibitor and phosphatase inhibitor 2 and 3 cocktails) to extract the protein. Equal amounts of protein (30 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membranes. Nonspecific proteins were blocked with 5% skim milk for 1 h at room temperature, and primary antibody was diluted in 1% BSA and reacted overnight at 4°C (dilution, 1:1,000). Then, the HRP-conjugated secondary antibody was reacted at room temperature for 1 h, and protein expression was measured using enhanced chemiluminescence (ECL) solution (GE Healthcare Life Science, Chicago, IL, United States). The density of the band was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, United States). The expression of proteins was normalized to that of actin.
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