To extract total protein from lenses, lenses were homogenized in lysis buffer composed of protease and phosphatase inhibitor cocktails for 1 h at 4 °C. The extracted protein concentration was quantified by BCA protein assay (Pierce), subjected to 7.5–10% SDS-Tris glycine gel electrophoresis, transferred to polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). The membrane was incubated in 5% non-fat skim milk in with Tween-20 buffer (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween-20, pH 7.4) for 1 h. Primary antibodies: AKR1B1, MMP9, vimentin (GTX113381, GTX100458 and GTX100619, respectively, Genetex, Irvine, CA, USA), N- and E-cadherin (610920 and 610182, respectively, BD Biosciences, Franklin Lakes, NJ, USA), Phosph-AMPK α1,2T172 (44-1150G, Thermo Fisher Scientific, Eugene, OR, USA), and SOD2 (acetyl K68) (ab137037, Abcam, Eugene, OR, USA) were diluted in PBST at 1:2000 dilutions and incubated at 4 °C overnight. Peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:10,000) were used. The result was visualized using ECL-Plus detection kit (GE Healthcare) and exposed to film. The developed films were scanned (photo scanner 4490, Epson, Long Beach, CA, USA), analyzed using NIH image densitometry analysis software (National Institutes of Health, Bethesda, MD, USA).
Ecl plus detection kit
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The ECL-Plus detection kit is a laboratory equipment product designed for chemiluminescent detection of proteins in Western blot analysis. It provides a sensitive and reliable method for visualizing and quantifying specific proteins within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by GE Healthcare (4 mentions)
Photo scanner 4490, by Epson (3 mentions)
Typhoon 9410 phosphorimager, by GE Healthcare (2 mentions)
Imagequant 5, by GE Healthcare (2 mentions)
Mouse anti vinculin, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Anti p akts473 4060, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Akr1b1, by GeneTex (1 mentions)
Gtx113381, by GeneTex (1 mentions)
Gtx100458, by GeneTex (1 mentions)
Gtx100619, by GeneTex (1 mentions)
Vimentin, by GeneTex (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Sc 2325, by Santa Cruz Biotechnology (1 mentions)
Amersham, by GE Healthcare (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
30 protocols using ecl plus detection kit
1
Lens Protein Extraction and Analysis
2
Quantitative Western Blot Analysis of Liver Proteins
3
Western Blotting Procedure
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Cell lysates were
prepared as described
above. The proteins were separated by SDS-PAGE and transferred onto
polyvinylidene difluoride membranes. The samples were heated at 95
°C for 10 min in 6× Laemmli loading buffer and run on SDS-PAGE
in buffer containing 25 mM Tris and 192 mM glycine (Bio-Rad, catalog
no. 4569033) at 120 V for 50 min. The membranes were blocked with
5% milk (Santa Cruz catalog no. SC-2325) in TBS-Tween (0.1% Tween)
for 1 h, incubated with primary antibody o.n. at 4 °C and then
washed in TBS-Tween (0.1%, TBST) two times for 10 min each. After
incubation with secondary antibodies conjugated to HRP, the blots
were washed three times in TBS-Tween (5 min each) and processed using
an ECL Plus detection kit (GE Healthcare, Amersham ECL Prime catalog
no. RPN2232) or the SuperSignal West Femto Maximum Sensitivity Substrate
(Thermo Scientific) as instructed by their suppliers.
prepared as described
above. The proteins were separated by SDS-PAGE and transferred onto
polyvinylidene difluoride membranes. The samples were heated at 95
°C for 10 min in 6× Laemmli loading buffer and run on SDS-PAGE
in buffer containing 25 mM Tris and 192 mM glycine (Bio-Rad, catalog
no. 4569033) at 120 V for 50 min. The membranes were blocked with
5% milk (Santa Cruz catalog no. SC-2325) in TBS-Tween (0.1% Tween)
for 1 h, incubated with primary antibody o.n. at 4 °C and then
washed in TBS-Tween (0.1%, TBST) two times for 10 min each. After
incubation with secondary antibodies conjugated to HRP, the blots
were washed three times in TBS-Tween (5 min each) and processed using
an ECL Plus detection kit (GE Healthcare, Amersham ECL Prime catalog
no. RPN2232) or the SuperSignal West Femto Maximum Sensitivity Substrate
(Thermo Scientific) as instructed by their suppliers.
4
VZV Glycoprotein H Interaction Studies
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Briefly, CHO-K1 Cre cells were transfected with gH expressing vectors and the pcDNA3.1-gL vector, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and lysates were harvested at 24 hours post transfection (hpt) [27] (link). Lysates from melanoma cells infected with recombinant VZV were harvested at 48 hours post infection (hpi). Immunoprecipitation and western blotting for gH used anti-gH monoclonal antibody SG3 (Meridian Life Science). Western blotting of viral proteins and cellular proteins was performed on infected-melanoma lysates as a control for infection and for sample loading. Membranes were probed for gE (mouse mAb 8612 anti-gE; Millipore), IE63 (rabbit anti-IE63; a kind gift of William Ruyechan, State University of New York, University at Buffalo, Buffalo, NY), and alpha-tubulin (clone B-5-1-2 mouse anti-α-tubulin; Sigma). All primary antibodies were detected using secondary horseradish peroxidase-conjugated antibodies to either anti-mouse or anti-rabbit and ECL Plus Detection Kit (GE Healthcare Bio-Sciences).
5
Western Blot Analysis of GABAergic Proteins
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The protein extracts were separated on 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (GE Healthcare, Buckinghamshire, UK). Western blotting49 (link) was performed using anti-GABAAR, anti-GAD1 and anti-GAD2 (Proteintech, Chicago, USA), anti-GABABR (Novus, Bangkok, Thailand), and anti-β-actin (Chemicon, Temecula, CA) antibodies. The membranes were subsequently incubated with an HRP-labeled goat anti-rabbit (or mouse) secondary antibody. The membranes were developed using an ECL Plus detection kit (GE Healthcare). The resulting bands were visualized and quantified using a Fuji Film Luminescent Image Analyzer (LAS-4000, Fuji Film Co., Tokyo, Japan) and analyzed using Multi Gauge Ver. 3.0 Image Analysis software (Fuji Film).
6
Monoclonal Anti-ABCB4 Antibody Protocol
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The monoclonal P3II-26 anti-ABCB4 antibody was obtained from Enzo Life Science (Villeurbanne, France). Rabbit polyclonal anti-EBP50 was from Thermo Fisher Scientific (Rockford, IL). Rabbit polyclonal anti-Flag was obtained from Abcam (Cambridge, UK) and the monoclonal anti-β actin from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Rabbit polyclonal anti-c-myc was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor-labeled secondary antibodies, DRAQ5 fluorescent probe and culture media were from Invitrogen-Life Technologies (Cergy-Pontoise, France) and peroxidase-conjugated secondary antibodies were from Rockland Immunochemicals (Gilbersville, PA). The siRNA-EBP50 duplex, the control siRNA and the ECL-Plus detection kit were from GE Healthcare France (Orsay, France). The transfection reagents Turbofect and Fugene HD were purchased from Fermentas France (Villebon-sur-Yvette, France) and Promega (Charbonnières, France), respectively. All other reagents were obtained from Sigma-Aldrich.
7
Immunoblotting of Lens Proteins
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Total protein extracts were prepared by homogenizing the lenses in a lysis buffer with protease and phosphatase inhibitor cocktails and then incubating for 1 h at 4 °C. The extracted proteins (assessed by BCA protein assay; Pierce) were subjected to 7.5–10% SDS-Tris glycine gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK).
The membrane was blocked in 5% non-fat skim milk using Tris-buffered saline/Tween-20 buffer (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween-20, pH 7.4, slightly alkali), followed by incubation in anti-p67-phox (07-502), anti-GLUT1 (ab115730), rat anti-GLUT5 (GTX12098) and human anti-GLUT5 (GTX83626) primary antibodies at 1:1000 dilutions at 4 °C overnight. Peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000 dilution) were used. Proteins were detected using an ECL-Plus detection kit from GE Healthcare and exposed to film. The developed films were scanned (photo scanner 4490, Epson, Long Beach, CA, USA) and analyzed using NIH image analysis software (National Institutes of Health, Bethesda, MD, USA).
The membrane was blocked in 5% non-fat skim milk using Tris-buffered saline/Tween-20 buffer (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween-20, pH 7.4, slightly alkali), followed by incubation in anti-p67-phox (07-502), anti-GLUT1 (ab115730), rat anti-GLUT5 (GTX12098) and human anti-GLUT5 (GTX83626) primary antibodies at 1:1000 dilutions at 4 °C overnight. Peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000 dilution) were used. Proteins were detected using an ECL-Plus detection kit from GE Healthcare and exposed to film. The developed films were scanned (photo scanner 4490, Epson, Long Beach, CA, USA) and analyzed using NIH image analysis software (National Institutes of Health, Bethesda, MD, USA).
8
Sodium Channel Protein Expression Assay
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Frozen ventricular tissue was lysed with a TissueLyser II (Qiagen) and protein concentration of the lysate was measured with the Bio-Rad Protein Assay. Ten to fifty micrograms of protein was loaded onto an 8–12% SDS polyacrylamide gel. After gel electrophoresis, proteins were transferred to a nitrocellulose membrane (0.2 μm, Bio-Rad Laboratories), which was blocked with 5% BSA. For blotting of the large (240 kDa) Nav1.5 protein, a gradient gel (4–15%, Bio-Rad Laboratories, #456–1,083) was used and protein samples transferred to a 0.4 μm membrane. Membranes were incubated with primary antibody, after which incubation with Cy5 (GE Healthcare, PA45012) or Cy3 (PA43010)-linked secondary antibody was performed and blots visualized with the ECL Plus Detection Kit (GE Healthcare) using a Typhoon 9400 scanner (GE Healthcare). The antibodies used were: Anti-Nav1.5 (Alomone labs, ASC-005), Anti-VEGF-B (R&D Systems, Af751), and β-actin (Cell Signaling Technology, 4967). Ponceau S staining solution (Sigma-Aldrich, St. Louis, MO, USA) was used as per manufacturer's instructions to visualize total membrane protein as a loading control for the blotted Nav1.5 protein.
9
Western Blot Analysis of Protein Samples
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Cells were lysed in lysis buffer (20 mM Tris pH 7.5, 0.5% Triton X-100, 150 mM NaCl, phosphatase inhibitor mix (Roche), protease inhibitor mix (Roche)) on ice and clarified by centrifugation. Cell extracts were resolved on SDS-PAGE and transferred onto PVDF membrane. After blocking with 5% skim milk in TBS-T (20 mM Tris pH 7.5, 150 mM NaCl, and 0.1% Tween20), the membranes were probed with antibodies. Anti-FLAG M2 (Sigma-Aldrich), anti-tubulin (Sigma-Aldrich), anti-MAP1B C-terminus (H130; Santa Cruz Biotechnology) and anti-GST-HRP (GE healthcare) were used as probes and detection was performed using the ECL plus detection kit (GE healthcare).
10
Immunoblotting of Recombinant COL17 Proteins
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Immunoblotting of recombinant proteins covering human COL17 was performed as described previously (36 (link)). Briefly, each sample was solubilized in Laemmli's sample buffer and applied to SDS-polyacrylamide gels and then transferred to a nitrocellulose membrane. The membrane was blocked for 1 h at room temperature in 3% skimmed milk in TBS and then incubated with 1:20 diluted mouse serum samples overnight at 4°C. Bound antibodies were visualized using 1:500 diluted HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch). Color was developed with 4-chloro-1-naphthol in the presence of H2O2. For the detection of antibodies to murine BP230, recombinant murine BP230 proteins were used as a substrate. The membranes were incubated with 1:20 diluted mouse serum samples overnight at 4°C. Bound antibodies were visualized using 1:5,000 diluted HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch). As a positive control, 1:5,000 HRP-conjugated mouse anti-His-tag mAb-HRP-DirecT (MBL, Nagoya, Japan) was used. The blots were detected using the ECL Plus Detection Kit (GE Healthcare, Fairfield, CT).
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