MG6 microglial cells or the L4-L5 spinal dorsal horn cells were collected after morphine or siRNA treatment. The specimens were lysed in lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, phosphatase and protease inhibitor cocktail) and mixed with Laemmli sample buffer. Proteins (30 μg) were loaded into each lane and separated by 7.5% or 12% SDS–PAGE gel. After transfer, the blots were incubated overnight at 4 °C with an anti-cPLA2 rabbit polyclonal antibody (1:1,000; Cell Signaling, #2832), anti-phospho cPLA2 rabbit polyclonal antibody (1:1,000; Cell Signaling, #2831), anti-P2X4R rabbit polyclonal antibody (1:2,000; Alomone, APR-002), anti-KCNMB3 mouse monoclonal antibody (1:2,000; NOVUS, NBP2–12916) or anti-β-actin mouse monoclonal antibody (1:5,000; Abcam, ab8226). The primary antibodies were diluted in Can Get Signal Solution 1 (Toyobo). After being washed, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:1,000; GE Healthcare) for 1 h at room temperature. The membrane-bound horseradish peroxidase-labelled antibodies were detected using SuperSignal West Femto (Life technologies) with an image analyzer (LAS-4000; Fuji Photo Film). The bands that were evaluated by apparent molecular size were quantified using the ImageJ 1.47 h software program (NIH).
Supersignal west femto
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan, Germany, Australia, Israel
SuperSignal West Femto is a chemiluminescent substrate designed for the detection of low-abundance proteins in Western blot analysis. It provides a high signal-to-noise ratio and can detect picogram levels of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (20 mentions)
Nitrocellulose membrane, by Bio-Rad (20 mentions)
Chemidoc imaging system, by Bio-Rad (18 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (18 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (18 mentions)
Protease inhibitor cocktail, by Merck Group (17 mentions)
Supersignal west pico, by Thermo Fisher Scientific (16 mentions)
Gapdh, by Cell Signaling Technology (12 mentions)
Chemi imager 4400 system, by Bio-Techne (11 mentions)
Chemidoc, by Bio-Rad (10 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Protease inhibitor cocktail, by Roche (9 mentions)
Complete protease inhibitor cocktail, by Roche (9 mentions)
β actin, by Merck Group (9 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (9 mentions)
Coomassie plus protein assay kit, by Thermo Fisher Scientific (9 mentions)
β actin, by Cell Signaling Technology (9 mentions)
Immobilon p, by Merck Group (8 mentions)
Ne per nuclear extraction kit, by Thermo Fisher Scientific (8 mentions)
Quantity one, by Bio-Rad (8 mentions)
427 protocols using supersignal west femto
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of BV2 Cells
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The previously described WB method was implemented to perform and analyze western blot membrane.[34 (link)
] Briefly, PB118‐treated BV2 cell lysates were collected, protein concentrations were measured using the Pierce BCA protein kit (Pierce 23 225) and an equal amount of proteins were applied to the Wb analysis. Proteins were separated by the electrophoresis using the 4–12% Bis‐Tris SDS‐PAGE Gel System (Invitrogen, Thermo Fisher Scientific), followed by membrane transfer to PVDF membranes (Invitrogen, Thermo Fischer scientific). Next, the membranes were blocked for 30 min with the Superblock (Thermo Scientific) and then incubated overnight with primary antibodies following the dilutions mentioned below. The membranes were then washed with TBST (four times) and incubated with appropriate secondary antibodies (1:10 000 dilution) at room temperature for 2 h. Then, membranes were incubated with Supersignal West Femto (Life Technologies), and the images for immunoreactivity were developed and visualized by the LICOR Odyssey Fc. Quantifications of proteins of interest were performed by Image J software.
] Briefly, PB118‐treated BV2 cell lysates were collected, protein concentrations were measured using the Pierce BCA protein kit (Pierce 23 225) and an equal amount of proteins were applied to the Wb analysis. Proteins were separated by the electrophoresis using the 4–12% Bis‐Tris SDS‐PAGE Gel System (Invitrogen, Thermo Fisher Scientific), followed by membrane transfer to PVDF membranes (Invitrogen, Thermo Fischer scientific). Next, the membranes were blocked for 30 min with the Superblock (Thermo Scientific) and then incubated overnight with primary antibodies following the dilutions mentioned below. The membranes were then washed with TBST (four times) and incubated with appropriate secondary antibodies (1:10 000 dilution) at room temperature for 2 h. Then, membranes were incubated with Supersignal West Femto (Life Technologies), and the images for immunoreactivity were developed and visualized by the LICOR Odyssey Fc. Quantifications of proteins of interest were performed by Image J software.
3
Immunoblotting Analysis of Cell Signaling
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Total cell extracts were obtained using NP-40 buffer (150 mM sodium chloride, 1 % NP-40, 50 mM Tris HCl pH 8) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Roche). The extracts were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (GE Healthcare). The proteins were detected with appropriate antibodies using the ECL detection system (GE Healthcare) or Super Signal West Femto (Life Technologies). Antibodies against the following proteins were used: Scd4 (Pineda), Caveolin-1 (Abcam), p-Caveolin-1 (Tyr14), Erk1/2, p-Erk1/2, il-1beta, and TNFalpha (all from Cell signaling).
4
Y-Receptor Protein Levels in Skeletal Muscle
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Y receptor protein levels in SkM were determined by immunoblotting. Protein lysates were subjected to deglycosylation under denaturing reaction conditions according to manufacturer’s instructions (P0704, New England Biolabs). Following deglycosylation, lysates were heated at 60°C and cooled to room temperature. 20 μg of protein was loaded in 10% SDS gels and transferred to PVDF membranes using Stain-Free technology (Bio-Rad). Membranes were incubated in primary antibodies Y1r, Y2r, and Y5r (NBP1-59008, NB100-56480, NB100-1538, respectively, Novus Biologicals) diluted (1:1000) in a chemiluminescent blocking buffer (blØkTM – CH, Millipore) overnight at 4°C. After the overnight incubation, membranes were washed with TBST before incubation with appropriate secondary antibody (anti-rabbit, Proteintech or anti-goat, Thermo Scientific) at a 1:25000 dilution in blocking buffer at room temperature. Membranes were visualized after incubation in SuperSignalTM West Femto (Thermo Scientific) on the Bio-Rad ChemiDoc MP.
5
Western Blot Analysis of hiPSC-CMs
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hiPSC-CMs were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (all from Sigma-Aldrich). Equal amounts of samples were mixed with non-reducing Laemmli sample buffer (BioRad, Hercules, CA, USA) and denatured at 96 °C for 5 min. Proteins were then separated on 10–12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Membranes were blocked with 5% non-fat dry milk powder in Tris-buffered saline. Primary antibodies used for western blotting were as follows: anti-tubulin (1/1000; T5168, Sigma-Aldrich), anti-caspase-3 (1/500; #9662, Cell Signaling Technology, Danvers, MA, USA), anti-cleaved caspase-3 (1/200; #9661, Cell Signaling Technology), anti-Bax (1/500; #5023, Cell Signaling Technology), anti-Bcl-2 (1/200; #4223, Cell Signaling Technology) and anti-phospho-Histone γ-H2AX (1/1000; 05-636-200, Merck Millipore). Secondary antibodies were anti-IgG rabbit (1/5000; P0448 Dako, Santa Clara, CA, USA) and anti-IgG mouse (1/5000; A9044, Sigma-Aldrich). Detection was carried out using peroxidase-conjugated antibodies and SuperSignalTM West Femto (ThermoFisher Scientific). Reactions were visualized using an Amershan Imager 600 (GE Healthcare, Chicago, IL, USA) and quantified with the ImageJ software (ver. 1.53t, NIH, Bethesda, MD, USA).
6
Western Blot Analysis of Smad2, YAP, and p-YAP
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Cells were lysed in RIPA buffer (1% NP40, 0,5% deoxycholate, 0,1% sodium dodecyl sulfate in Tris-buffered saline (TBS)) (Sigma-Aldrich) supplemented with protease (Complete, Sigma-Aldrich) and phosphatase (PhosSTOP, Sigma-Aldrich) inhibitors. Equal amounts of samples were mixed with non-reducing Laemmli sample buffer (BioRad, Hercules, CA, USA) and denatured at 96 °C for 5 min. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat dry milk powder in TBS. Primary antibodies used for western blotting were: anti-Smad2 (1/500, Abcam, Cambridge, UK, ab228765), anti-YAP (1/1000, Cell Signaling Technology, Danvers, MA, USA, D8H1X), anti-phospho-YAP (1/1000, Cell Signaling Technology, D9W2I) and anti-GAPDH (1/1000, Cell Signaling Technology, D16H11). Secondary antibody was anti-IgG rabbit (1/5000, Dako, Santa Clara, CA, USA, P0448). Detection was carried out using peroxidase-conjugated antibodies and SuperSignalTM West Femto (Thermo Fisher Scientific). Reactions were visualized using an Amershan Imager 600 (GE Healthcare, Chicago, IL, USA) and quantified with ImageJ software (NIH, Bethesda, MD, USA).
7
Western Blot Quantification of PROS1 and SLC3A2
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For cell lysates approximately 10 μg and for brain lysates 50–60 μg of protein was loaded on a 12% SDS-polyacrylamide gel with 2,2,2-trichloroethanol (TCE) which allow detection of total protein load (Ladner et al., 2004 (link)). Proteins were transferred onto a PVDF membrane (Bio-Rad). Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C. Membranes were washed with TBS-Tween20 (0.1%) and incubated with an HRP-conjugated secondary goat anti-rabbit IgG antibody (Dako, P0448) for 2 h at room temperature. The membranes were washed three times with TBS-Tween (0.1%) and once with TBS, incubated with SuperSignalTM West Femto (Thermo Fisher Scientific) and imaged (Odyssey® Fc, LI-COR). Protein expression was corrected for total amount of protein determined by Gel DocTM EZ System (Bio-Rad).
8
Western Blot Membrane Processing
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PVDF membrane were retrieved after transfer and washed once with distilled water. The membrane was blocked at room temperature for 1 h in blocking buffer (5% skim milk in 0.1% PBS-Tween 20). The membrane was incubated overnight with the primary antibody (1:1000 in 5% BSA in 0.1% PBS-Tween 20) at 4°C in 50 ml plastic tubes on a tube roller. The membrane was washed five times for 5 min with 0.1% PBS-Tween 20 before being incubated for 1 h at room temperature with goat ant-imouse IgG H&L secondary antibody (1:2000 in 5% skim milk in 0.1% PBS-Tween 20). Finally, membranes were washed five times for 5 min with 0.1% PBS-Tween 20 before being treated with SuperSignalTM West Femto (Thermo-Fisher Scientific, UK). The chemiluminescent signal was captured with the Peqlab camera (VWR, UK) and analysed using Fusion molecular imaging software v 15.5 (Fisher Scientific, UK).
9
Zebrafish Cardiac Regeneration Protein Profiling
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Zebrafish amputated and control-sham ventricles were dissected at 3, 7, and 28 dpa. Pooled protein extracts were obtained from 2–4 ventricles for each experimental group, first by tissue mechanical homogenization in RIPA buffer (10 mM Tris pH 7.2, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X 100) and then by sonication, and thus considered as a single biological replicate. Denatured samples (5 min, 95 °C, 5% β-mercaptoethanol) were then separated by SDS-PAGE and transferred to PVDF-membranes following standard protocols. Antibodies used for these experiments were anti-LC3 (1:1000; rabbit, #2775S, Cell Signaling, USA), anti-Beclin1 (1:1,000; rabbit, #3738, Cell Signaling, USA), anti-GAPDH (1:5000; goat, #G9545, Merck. Germany), anti-4E-BP1 (1:1000; rabbit, #9644, Cell Signaling, USA), anti-phospho-4E-BP1 (Thr37/46) (1:1000; #2855, Cell Signaling, USA). The enhanced chemiluminescent substrate SuperSignalTM West Femto (Thermo Fisher Scientific, USA) was used for signal detection. Signal intensities were quantified using the software ImageJ77 (link), then normalized to their respective loading control (GAPDH), and finally to the signal corresponding to the sham-control condition.
10
Western Blot Analysis of Inflammatory Proteins
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Following colonic crypts homogenization, as described above, total protein was extracted by lysing tissue in ice-cold RIPA buffer supplemented with protease inhibitor cocktail (Santa Cruz #sc-24948), sodium orthovanadate, and PMSF. Protein concentrations were quantified using the BCA method. For each group, 15 μg of protein were transferred to a nitrocellulose membrane after electrophoretic separation. The membranes were blocked using 5% of either dry milk or BSA in Tris Buffered Saline + Tween20 (TBST) buffer. The nitrocellulose membrane was then incubated overnight with the following primary antibodies: cleaved GSDMD (Cell Signaling Technology, #50928), cleaved caspase-11 (Abcam, #ab180673), cleaved caspase-1 (Invitrogen #AB 5B10), and β-actin (BD Biosciences #612656) at 1:1000 dilution. After washing steps in TBST, the membrane was incubated at room temperature for 1 h with anti-rabbit IgG (H + L) or anti-mouse IgG (H + L) (both Jackson ImmunoResearch) at 1:15,000 dilution. Proteins were visualized using SuperSignalTM West Femto or SuperSignal West Pico PLUS (both Thermo Fisher) chemiluminescent detection kits.
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