Immunoblotting was performed as described previously21 (link). The anti-KAP3, anti-laminin, and anti-β-actin antibodies were used for immunoblotting at dilutions of 1:500, 1:400, and 1:2000, respectively. For immunodetection, anti-rabbit IgG or anti-mouse IgG antibody (Cell Signaling Technology, Beverly, MA, USA) was used as the secondary antibody at a dilution of 1:5000 or 1:10,000, respectively. Antibody binding was detected using the Enhanced ChemiLuminescence (ECL) system (GE Healthcare, Chicago, IL, USA).
Enhanced chemiluminescence ecl system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Enhanced chemiluminescence (ECL) system is a laboratory equipment designed to detect and quantify proteins in Western blot analyses. It utilizes a chemiluminescent reaction to produce light, which is then captured and measured by a compatible imaging device. The system provides a sensitive and reliable method for protein detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (3 mentions)
Sch772984, by Selleck Chemicals (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Quantity one software v4, by Bio-Rad (2 mentions)
Anti mouse igg antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Novex sharp standard, by Thermo Fisher Scientific (1 mentions)
Kras 12063 1 ap, by Proteintech (1 mentions)
Vemurafenib s1267, by Selleck Chemicals (1 mentions)
Dylight680, by Cell Signaling Technology (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Merck Group (1 mentions)
β actin a5441, by Merck Group (1 mentions)
Dylight800, by Cell Signaling Technology (1 mentions)
Tubulin t 9026, by Merck Group (1 mentions)
Zstk474, by Selleck Chemicals (1 mentions)
Abt 263, by Santa Cruz Biotechnology (1 mentions)
Perk 3192, by Cell Signaling Technology (1 mentions)
20 protocols using enhanced chemiluminescence ecl system
1
Immunoblotting of KAP3, Laminin, and β-Actin
2
Microvesicle Protein Analysis by Western Blot
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Microvesicle lysates were prepared as described above. 26 μl of each sample was loaded to a 4–12 % Bis-Tris Gel (NuPAGE, Life Technologies). Novex® Sharp Standard (Life technologies) was used as a standard. The gel was run for 1 h at 175 V in MES buffer (NuPAGE, Life Technologies) followed by transfer for 1 h at 20 V onto a PVDF membrane (Invitrogen). The membrane was blocked with 5 % BSA in TBS with 0.2 % Tween (TBS-T) for 1 h (RT), followed by washes in TBS-T and incubation with Flotillin-1 (mouse monoclonal, 1:500, BD Biosciences) antibody in 0.5 % BSA in TBS-T over night (4 °C). After extensive washes in TBS-T the membrane was incubated with a peroxidase-conjugated goat anti-mouse IgG (1:20 000, Pierce) antibody in 0.5 % BSA in TBS-T for 1 h (RT) and then finally washed again in TBS-T. The enhanced chemiluminescence (ECL) system (GE Healthcare) was used for development.
3
DMEM-Based Cell Culture Protocol
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Dulbecco's Modified Eagle's Medium (DMEM), FBS, Penicillin/Streptomycin and Glutamine were all purchased from Life Technologies. BIRB796 (S1574), CB1954 (S7829), PLX-4072 (S1267), Vemurafenib (S1267) and ZSTK474 (S1072) were purchased from Selleckchem; Trametinib (HY-10999) was from Insight Biotechnologies; JNK inhibitor VIII (CAS 894804-07-0) was from Calbiochem; SCH772984 (S7101) was from Merck. Bovine serum albumin (BSA), DMSO and Doxycycline were purchased from Sigma. Vectashield (H-1200-10) was purchased from VectorLabs. The following antibodies were all purchased from Cell Signalling Technologies: p-S473 AKT (4060), Akt1 (2967), c-Jun (9165), p-T202/Y204 ERK1/2 (4370), ERK1/2 (9107), GFP (2955), p-S217/S221 MEK1/2 (9154), MEK1/2 (4694), p-T334 MAPKAPK2 (8753), MAPKAPK2 (3042). BRAF (3967S) was purchased from Santa Cruz. Tubulin (T9026) and β-actin (A5441) were purchased from Sigma and KRAS (12063-1-AP) was from ProteinTech. Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad and the enhanced chemiluminescence (ECL) system from GE Healthcare was used for detection. The Dylight™680 and Dylight™800 conjugated secondary antibodies were from Cell Signalling Technology.
4
Investigating Cell Stress Signaling Pathways
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Thapsigargin was purchased from Invitrogen. Tunicamycin was purchased from Enzo Life Sciences. 4-hydroxytamoxifen (4-HT) was purchased from Sigma; in all experiments involving 4-HT 0.1% ethanol, the diluent for 4-HT, was used and was without effect. Selumetinib (AZD6244/ARRY-142886) and AZD8055 were provided by Paul Smith, AstraZeneca, Alderley Park, Macclesfield, UK, whilst SCH772984 was purchased from Selleck Chemicals. ABT-263 was purchased from Santa Cruz Biotechnology. GSK2606414 and QVD-oPh were purchased from MERCK and Calbiochem respectively. Antibodies specific for BCLXL (2762), BiP (3177), CHOP (2895), eIF2α (9722), p-eIF2α (Ser51) (9721), p-ERK1/2 (Thr202/Tyr204) (9106), IRE1α (3294), PARP (9542) and PERK (3192) were purchased from Cell Signaling Technology; ERK1 (610031) from BD Biosciences; BIM (AB17003) from Millipore; BMF (ALX-804-343) from Enzo Life Sciences; PUMA (3043) from ProSci; BAK (sc-832), BAX (sc-493), BCL2 (sc-7382) and MCL1 (sc-819) from Santa Cruz Biotechnology; Atg5 (A0731) and β-Actin (A544) from Sigma. Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad and the enhanced chemiluminescence (ECL) system from GE Healthcare was used for detection.
5
Western Blot Analysis of p65 NF-κB
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Fifty micrograms of lung lysates were separated with 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and further transferred to a nylon membrane, which was incubated with p65 nuclear factor (NF)-κB primary antibody (sc-71675) (1:1500 dilution, 4°C, overnight) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), then incubated with peroxidase–conjugated secondary antibody (1:1000 dilution, at room temperature, 1 h) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and developed with an enhanced chemiluminescence (ECL) system (GE Healthcare Life Sciences, Chalfont, U.K.). GAPDH (sc-32233) was used to normalize.
6
Protein Extraction and Western Blot Analysis
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Cell lysates were extracted by a modified RIPA buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris (pH 7.4), 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail) (Sigma-Aldrich, Burlington, MA, USA). After sonication, total protein (20 μg) was segregated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Protech, Taipei, Taiwan) and transferred to a polyvinylidene difluoride membrane (Merck, Darmstadt, Germany). The membrane was blocked using 5% non-fat dry milk (Anchor, Auckland, New Zealand) for 1 h at room temperature, hybridized with primary antibody overnight at 4 °C, and washed by PBST, before being incubated with horseradish peroxidase-conjugated secondary antibody (Invitrogen, Waltham, MA, USA) for 2 h at room temperature. An enhanced chemiluminescence (ECL) system (GE Healthcare Life Sciences, Buckinghamshire, UK) was used to detect the target proteins on Western blots. Band intensity was quantified using ImageJ software. The concentrations and product codes of the primary and secondary antibodies used for Western blotting in this study are listed in Table S3 .
7
Investigating Cellular Stress Responses
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Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), and other cell culture products were purchased from Life Technologies (Grand Island, NY, USA). Lipopolysaccharide (LPS), 4-phenylbutyric acid (4-PBA), salubrinal, and 2,3-diaminonaphthalene (2,3-DAN) were obtained from Sigma-Aldrich (St. Louis, MO, USA). TRIzol reagent and lipofectamine siRNA transfection reagent were purchased from Invitrogen (Carlsbad, CA, USA). Small interfering RNAs (siRNAs) against scrambled control and CHOP were obtained from M Biotech (Seoul, Korea). Antibodies against COX-2, CHOP, and ATF6α (p90) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-PERK (p-PERK), nuclear factor κB (NFκB), and iNOS were purchased from BioLegend (San Diego, CA, USA). Antibodies against phosphor-IRE1α were purchased from Abcam (Cambridge, MA, USA). Antibodies against phospho-IκB (p-IκB) and IκB were purchased from Cell Signaling Technology (Beverly, MA, USA). An enhanced chemiluminescence (ECL) system was obtained from GE Healthcare Life Sciences (Chicago, Illinois, USA).
8
Western Blot Analysis of GR and Associated Proteins
9
Nitrotyrosine Quantification by Slot-Blot
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Relative amounts of proteins nitrated on tyrosine were measured by use of slot-blot techniques. Assay lysate was transferred onto nitrocellulose membrane using a slot-blot microfiltration unit (Bio-Rad Laboratories, Hercules, CA). Nitrotyrosine was detected by use of a mouse monoclonal anti-nitrotyrosine antibody (Cayman Chemical, Ann Harper, MI) followed by followed by horseradish peroxidase-conjugated secondary antibody (1∶2000, GE Healthcare Bio-Sciences, Pittsburgh, PA). Immunoreactive proteins were detected using the enhanced chemiluminescence (ECL) system (GE Healthcare Bio-Sciences, Pittsburgh, PA). Relative levels of nitrotyrosine immunoreactivity were determined by Image J software.
10
Western Blot Analysis of Phospho-ERK1/2
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Equal protein amounts of cell lysate samples were electrophoresed through reducing sodium dodecyl sulfate (SDS) polyacrylamide gels and electroblotting onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) [3 (link)]. Membranes were then blocked with 5% bovine serum albumin and incubated with primary antibodies for phospho-ERK1/2 (Thr202/Tyr204; Catalog # 4370; Cell Signaling Technology, Danvers, MA, USA) or ERK1/2 protein (Catalog # 9102; Cell Signaling). Membranes were washed and incubated with horseradish peroxidase-linked secondary antibodies, and Enhanced Chemiluminescence (ECL) System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) was used for detection. Autoradiography was performed using UltraCruz Autoradiography Films (Santa Cruz Biotechnology, Dallas, TX, USA), and optical densities of protein bands were quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
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