Fetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Gibco (Grand Island, NY, USA). Penicillin-streptomycin and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Hyclone (Logan, UT, USA). Dextrose, sodium bicarbonate, 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bicinchoninic acid (BCA) kit and SuperSignalTM west Femto chemiluminescent substrate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against Smad2/3 and kidney injury molecule-1 (KIM-1) were purchased from Cell Signaling (Denver, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore (Temecula, CA, USA). Antibodies against AhR, hypoxia-inducible factor 1-alpha (HIF-1α), epithelial cadherin (E-cad), fibronectin (FN), and Bcl-2-associated X protein (Bax), caspase-3 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). AhR siRNA (5′-GUGACUUGUACAGCAUAAUTT-3′) was purchased from GenePharma (Shanghai, China), Smad2/3 siRNA (sc-37238) was purchased from Santacruz Biotechnology (Santacruz, CA, USA), and HIF-1α siRNA (SASI_HS02_00332063) was purchased Sigma-Aldrich (St. Louis, MO, USA).
Supersignal west femto chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Japan, Denmark
SuperSignal West Femto Chemiluminescent Substrate is a laboratory product designed to detect low-abundance proteins through chemiluminescence. It provides high sensitivity for western blot applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (18 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (18 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (14 mentions)
Protease inhibitor cocktail, by Merck Group (13 mentions)
Nitrocellulose membrane, by Bio-Rad (11 mentions)
Quantity one software, by Bio-Rad (11 mentions)
Protease inhibitor cocktail, by Roche (10 mentions)
Bca protein assay, by Thermo Fisher Scientific (10 mentions)
C digit blot scanner, by LI COR (9 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (9 mentions)
Bis tris gel, by Thermo Fisher Scientific (8 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (8 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Chemidoc xrs system, by Bio-Rad (7 mentions)
Dc protein assay, by Bio-Rad (7 mentions)
Protease inhibitor, by Merck Group (7 mentions)
Gel doc xr system, by Bio-Rad (6 mentions)
Ripa buffer, by Santa Cruz Biotechnology (6 mentions)
Ripa lysis buffer, by Merck Group (6 mentions)
315 protocols using supersignal west femto chemiluminescent substrate
1
Investigating Cellular Pathways and Toxicity
2
Quantifying AKR1B1 Protein Expression
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The proteins were isolated from the cells via homogenates using a radio-immuno-precipitation assay (RIPA, Sigma, MO, United States). Western blotting was implemented based on the standard procedure as previously described (Wang et al., 2018b (link)). Primary antibodies were used to detect the expression of AKR1B1 (1:1500, Proteintech, 15439-1-AP, Wuhan Sanying). The loading control was a β-actin antibody (1:1000, Abcam, ab32572, New Territories, HK). After incubation with secondary antibodies (HRP-conjugated goat anti-rabbit IgG 1:4000, EarthOx, 7074S, Millbrae, United States), the samples were developed with SuperSignalTM West Femto Chemiluminescent Substrate (ThermoFisher, Rockford, United States) and the Gel Doc™ XR + System (Bio-Rad, CA, United States).
3
Protein Extraction and Western Blot Analysis
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Proteins derived from the femoral head of the mouse embryos and ATDC5 cells were isolated using a radio-immuno-precipitation assay buffer (RIPA, Sigma–Aldrich, St. Louis, MO, USA). The concentration of protein was quantified with a BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). The extracted protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA). The membrane was blocked with 5% nonfat milk and then incubated with Bmp2 (1:500, Santa Cruz Biotechnology, sc137087, USA), Phospho-p38 (1:1000, Cell Signaling Technology, #9211, USA), p65 (1:1000, Cell Signaling Technology, SA) and β-actin (1:3000; Proteintech, 60008-1-1 g, Rosemont, USA) in a TBST buffer at 4 °C overnight. After incubation with the secondary antibody, either HRP goat anti-rabbit IgG (1:3000; EarthOx, 7074 S, Millbrae, USA) or HRP goat anti-mouse IgG (1:3000; EarthOx, 7076 S, Millbrae, USA), the samples were developed with SuperSignalTM West Femto Chemiluminescent Substrate (ThermoFisher, Rockford, USA) and the Gel Doc™ XR + System (Bio-Rad, CA, USA). Quantity One (Bio-Rad, Hercules, CA, USA) was used to capture the chemiluminescent signals and analyze the data. All samples were performed in triplicate.
4
Quantification of Nkx2.5 Protein Levels
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Western blotting was performed in accordance with a standard procedure using a polyclonal antibody that specifically recognizes Nkx2.5. The collected hearts from E13.5, E15.5 and E18.5 developing embryos were frozen in liquid nitrogen and kept at -80°C. The protein from the hearts or H9c2 cells was isolated from tissue homogenates or cell lysate using a radio-immuno-precipitation assay (Sigma, St. Louis, MO, USA) buffer that was supplemented with protease and phosphatase inhibitors. The protein concentrations were quantified using the BCA assay. The extracted protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% nonfat milk and then incubated with anti-Nkx2.5 antibody (1:1000, Abcam, New Territories, HK) in TBS buffer at 4°C overnight. The loading control was β-actin antibody (1:3000, Santa, Santa Cruz, CA, USA). After incubation with the secondary antibody HRP goat anti-rabbit IgG (1:3000, EarthOx, Millbrae, CA, USA), the blots were developed with SuperSignalTM West Femto Chemiluminescent Substrate (ThermoFisher, Waltham, MA, USA), and the Gel Doc™ XR+ System (Bio-Rad, Hercules, CA, USA) and Quantity One software were used to capture the chemiluminescent signals.
5
Western Blotting for Phosphorylated Signaling Proteins
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Cells cultured under the conditions indicated were harvested, washed with ice-cold PBS, and lysed on ice for 15−30 min in a lysis buffer (20 mM Tris, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, and 1 µg/ml aprotinin and leupeptin). Cell lysates were resolved by 4−12% Bis-Tris SDS-PAGE Gel (Invitrogen), transferred onto nitrocellulose membrane (Invitrogen), blocked with 5% dry non-fat milk in Tris buffered saline (pH 7.4) containing 0.1% Tween-20, and probed with the following Abs to (Abs were used at 1:1000 and purchased from Cell Signaling Technology unless otherwise described): p-STAT3 (Tyr705; Cell Signaling Technology), p-SMAD2 (Ser465/467; Cell Signaling Technology), p-SMAD3 (Ser423/425; Cell Signaling Technology), and β-actin (Sigma-Aldrich; 1:10,000). Immunoreactivity was detected by SuperSignalTM West Pico Chemiluminescent Substrate (Thermo Scientific) and SuperSignalTM West Femto Chemiluminescent Substrate (Thermo Scientific) with LAS-3000 (FujiFilm).
6
Western Blot Immunodetection Protocol
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Protein samples either from cells were loaded in SDS-PAGE gel in electrophoresis buffer (2.5 mM Tris-base, 19.2 mM Glycine and 0.1% SDS) at 120 V for 2 h. Proteins were transferred to the PVDF membrane in transfer buffer (2.5 mM Tris- base, 19.2 mM Glycine and 20% MeOH) at 100 V for 1 hour 30 min. The membrane was blocked in TBST (100 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.5) containing 5% skim milk for 1 hour and incubated at 4 °C overnight with antibodies including α-c-Myc produced in rabbit (Sigma-Aldrich, C3956, 1:2,000), and α-HA high affinity produced in rat (Sigma-Aldrich, 11867423001, 1:1,000). After washing with TBST, the membranes were incubated with a peroxidase-AffiniPure Donkey α-Rabbit IgG (H+L) (Jackson ImmunoResearch, 711–035-152, 1:5,000) or Peroxidase AffiniPure Goat Anti-Rat IgG (H+L) (Jackson ImmunoResearch, 112–035-003, 1:10,000) at RT for 1 hour. For exposure, the membrane was rinsed with TBST and was subjected to SuperSignal TM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, PI34580) or SuperSignalTM West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, PI34095) and analyzed on a ChemiDocTM XRS+ system (Bio-Rad).
7
Tyrosine Hydroxylase Protein Quantification
8
Activin A Protein Detection by Western Blot
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SDS-PAGE gels (13.5%) were run with a constant current of 30 mA and a voltage ceiling of 160V, and then transferred to a PVDF membrane for 1h at 100V. Membranes were blocked with 5% nonfat dry milk in TRIS-buffered saline with 0.1% Tween 20. Protein was detected with a primary mouse monoclonal anti-human activin A ab (clone 69403, R&D Systems) and a secondary HRP-conjugated goat anti-mouse ab (Life Technologies, Grand Island, NY, USA). Imaging was then performed with SuperSignal™ West Femto chemiluminescent substrate (Life Technologies) on an Alpha Innotech FluoroChem™ HD imager.
9
Activin A Protein Detection by Western Blot
10
Western Blot Analysis of STING Pathway
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Cells were lysed in Cold Spring Harbor NP-40 lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, and 1.0% Nonidet-P40) supplemented with protease and phosphatase inhibitors (PMSF, NaF, Na3VO4, and Roche PhosSTOP) and then centrifuged at 12,000 g for 15 min at 4°C to get cellular lysate. Equal amounts of protein were loaded and size separated on an SDS–PAGE gel and wet transferred onto polyvinylidene fluoride membrane. The membrane was blocked in tris-buffered saline and Tween 20 (TBS-T) buffer with 5% milk for 1 h at room temperature, followed by overnight incubation with primary antibodies diluted in TBS-T with 5% BSA. Membrane was washed three times with TBS-T buffer for 10 min, incubated at room temperature with HRP-conjugated IgG secondary antibody (Jackson Immunoresearch), washed three times with TBS-T for 10 min followed once with TBS buffer for 10 min, and then developed with SuperSignal West Femto Chemiluminescent Substrate (Life Technologies).
Rabbit antibodies against TBK1 (D1B4), pTBK1 (Ser172, D52C2), STING (D2P2F), p-STING (Ser365, D8F4W), p-STING (Ser366, D7C3S), FLAG (D6W5B), HA (C29F4), and GFP (D5.1) were from Cell Signaling Technology. Rabbit antibody against SURF4 was from Novus. Mouse antibodies against GAPDH and β-Tubulin were from Santa Cruz Biotechnology. Mouse antibody against GM130 was from BD Biosciences.
Rabbit antibodies against TBK1 (D1B4), pTBK1 (Ser172, D52C2), STING (D2P2F), p-STING (Ser365, D8F4W), p-STING (Ser366, D7C3S), FLAG (D6W5B), HA (C29F4), and GFP (D5.1) were from Cell Signaling Technology. Rabbit antibody against SURF4 was from Novus. Mouse antibodies against GAPDH and β-Tubulin were from Santa Cruz Biotechnology. Mouse antibody against GM130 was from BD Biosciences.
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