Untransfected HeLa, 5637, 1321, A172, NCCIT and NT2 cell cultures were lysed with 1 ml of lysis buffer [1% Triton X-100, 5 mM EDTA, 50 mM TrisHC1, pH=7.4, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS)], 1% protease inhibitor cocktail and phosphatase inhibitor (Sigma, USA). Protein concentrations were measured by the Bradford Protein Assay reagent. Briefly, 20 µg of cell lysates were isolated on 12.5% SDS-PAGE and transferred onto a Polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Blocking was carried out with 5% skim milk in tris-buffered saline (Sigma-Aldrich, Germany), containing 0.05% Tween20 (TBS-T). The membrane was then incubated for 1 hour with primary mouse anti-Oct-3/4 sc-5279 antibody (Santa Cruz, USA) diluted in 3% blocking buffer, and washed with PBS for 30 minutes at room temperature. A secondary HRPconjugated sheep-anti-mouse antibody (Avicenna Research Institute, Iran) was added next and incubated at RT for 1 hour. After washing, signals were detected using the Amersham ECL Prime Western Blotting Detection kit (GE Healthcare Life Sciences) and quantified by Gel Logic 2200 (Kodak, Japan).
Amersham ecl prime western blotting detection kit
Manufactured by GE Healthcare
Sourced in United Kingdom
The Amersham ECL Prime Western Blotting Detection kit is a laboratory reagent used for the visualization and quantification of proteins on Western blots. The kit contains the necessary components to detect and analyze target proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Amersham ecl prime, by Cytiva (9 mentions)
Primary mouse anti oct 3 4 sc 5279 antibody, by Santa Cruz Biotechnology (2 mentions)
Gel logic 2200, by Kodak (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Tris buffered saline (tbs), by Merck Group (1 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Santa Cruz Biotechnology (1 mentions)
Mouse anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Mouse anti icam 1, by Santa Cruz Biotechnology (1 mentions)
Chemidoc mp system software, by Bio-Rad (1 mentions)
Rabbit anti e selectin, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti vcam 1, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Pall Corporation (1 mentions)
Image lab software 6, by Bio-Rad (1 mentions)
Ab9400, by Abcam (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Ac 15, by Merck Group (1 mentions)
Ab39072, by Abcam (1 mentions)
9 protocols using amersham ecl prime western blotting detection kit
1
Characterization of Oct-3/4 Expression
2
Polyclonal Antibody Production and Validation
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Polyclonal antibodies against purified Lpg0393 was raised by injecting 30 μg of the purified protein into a male albino rabbit with complete Freund’s adjuvant and giving two booster injections with the same amount of the protein 7 and 14 days later. For immunoblotting, GST-tagged Rab proteins and Lpg0393 were incubated with GST-Bind Resin for 1 h at 4°C, and the resin was washed four times with 200 μl of Buffer A. Resin-bound proteins were separated on a 15% SDS-PAGE gel and transferred electrophoretically to a Immobilon-P PVDF membrane (Millipore). The membrane was blocked by immersion in the PBST buffer containing 5% skimmed milk at room temperature for 4 h, and then transferred to a PBST buffer containing the antiserum against Lpg0393 (1:500 dilution). After incubation at 4°C for 12 h, the membrane was incubated with anti-rabbit secondary antibody (Cell Signaling) at room temperature for 1 h and visualized using Amersham ECL Prime Western blotting detection kit (GE Healthcare).
3
Western Blot Protein Extraction and Detection
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Human OFs were removed from the incubator and placed on ice. Cells were then washed three times with ice-cold PBS and lysed for 30 min with RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 100 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 mM Na3VO4, and 1X Complete™ Protease Inhibitor Cocktail (Santa Cruz Biotechnology)]. Equal amounts of protein extracts were loaded onto 10–15% SDS-PAGE gels, electrophoresed, and transferred to PVDF membranes (Millipore, Bedford, MA). Membranes were blocked in Tris-buffered saline with 0.05% Tween-20 (TBST) supplemented with 5% powdered milk or 5% bovine serum albumin, and then incubated with a primary antibody against the designated protein. Blots were then washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody in TBST with 5% powdered milk. Bound antibodies were detected with the Amersham ECL Prime Western Blotting Detection kit (GE Healthcare, Buckinghamshire, UK).
4
Quantifying Cell Adhesion Molecules
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Whole‐cell lysates were prepared in modified RIPA buffer (50 mmol/L of HEPES, 20 mmol/L of pyrophosphate, 25 mmol/L of β‐glycerophosphate, 50 mmol/L of NaF, 5 mmol/L of Na2MoO4, 5 mmol/L of EDTA, 150 mmol/L of orthophenanthrol, 1% NP‐40, 2% deoxycholate, and 1% Triton X‐100) in the presence of protease and phosphatase inhibitors. Protein concentrations were quantified using the Pierce BCA Protein Assay (Thermo Scientific, Waltham, MA). Samples (25 μg protein) were denatured, resolved on an 8% SDS‐polyacrylamide gel, and then transferred to an Immuno‐blot PVDF membrane (Amersham Biosciences, Piscataway, NJ). Incubation with primary antibodies (rabbit anti‐VCAM‐1, mouse anti‐ICAM‐1, rabbit anti‐E‐Selectin, and mouse anti‐GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA) was performed for 2 hours, followed by HRP‐linked anti‐rabbit IgG or anti‐mouse IgG (Bio‐Rad, Hercules, CA) secondary antibodies for 40 minutes at room temperature. Immunoreactive bands were visualized by chemiluminescence using an Amersham ECL Prime Western Blotting Detection Kit (GE Healthcare, Pittsburgh, PA). Densitometry was performed using ChemiDoc MP System software (Bio‐Rad).
5
Immunoblotting Validation of Differentially Expressed Proteins in PCOS
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Validation of differentially expressed proteins was carried out by immunoblotting. 15 μg of follicular fluid protein from 7 women with PCOS and 7 controls were resolved by 10% SDS-PAGE and trans-blotted onto a PVDF membrane (Pall corporation, Pensacola, Fl, USA). The membranes were blocked with 5% non-fat dry milk followed by incubation for 16 hours with mouse anti-human SERPINA1 antibody (1:10000, ab9400 Abcam, MA, USA) or goat anti-human ITIH4 (1:500, ab92338 Abcam, MA USA), respectively. After incubation, blots were washed with PBS, incubated with secondary antibody, horseradish peroxidase (HRP)-labelled anti-mouse (1:10000) or anti-goat (1:10000) antibody, (Dako, CA, USA), and incubated for 1 hour. Detection was carried out using ECL prime detection reagents (Amersham ECL Prime western blotting detection kit, GE Healthcare, UK). The band intensities were quantified using Image Lab Software 6.0.1 (Bio-rad, USA) against Coomassie Brilliant Blue (CBB) stained total loading control.
6
Protein Extraction and Western Blot Analysis
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Proteins were lysed from the cells and the tissues using lysis buffer containing 20 mmol/L Tris‐Cl (pH 8.0), 1% Triton X‐100, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.05% sodium dodecyl sulfate (SDS), 1% Na‐deoxycholate, and fresh 1× protease inhibitors. The concentration of each protein was measured by the DC Protein Assay kit (Bio‐Rad Laboratories, Hercules, CA) before the proteins were equally loaded and separated by SDS‐polyacrylamide gel electrophoresis. Proteins were then transferred to FluoroTrans‐W® membranes (Pall, Port Washington, NY) and incubated overnight with primary antibodies against GLP‐1R (ab39072, Abcam; 1:1000), Adrβ3 (ab94508, Abcam; 1:1000), and β‐actin (1:1000; AC‐15, Sigma‐Aldrich). The membranes were then treated with the horseradish peroxidase–conjugated secondary antibody at a 1:10 000 to 15 000 dilution. The Amersham ECL Prime Western Blotting Detection kit (GE Healthcare, Freiburg, Germany) was used for the determination of targeted proteins. Protein levels quantitated from Western blots were normalized by loading β‐actin.
7
Western Blot Analysis of Protein Markers
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The isolated hippocampus and cortex samples were homogenized in buffer containing 20 mM Tris-HCl, pH 7.0, 6 M urea, 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100. The homogenates were subjected to 8% SDS-polyacrylamide gel electrophoresis and analyzed by Western blot using rabbit anti-TCTP (diluted 1 : 1000, Abcam, Cambridge, MA), rabbit anti-SUCLA2 (diluted 1 : 1000, Abcam), rabbit anti-NSE (diluted 1 : 1000, Abcam), and rabbit anti-GAPDH (diluted 1 : 10,000, AbFrontier, Seoul, Korea) antibodies at 4°C overnight. The membranes were incubated with the indicated secondary antibody (diluted 1 : 5000, GE Healthcare, Madison, WI). All values were corrected with reference to the value for GAPDH, used as an internal standard. Immunoreactivity was detected by using an Amersham ECL Prime Western blotting detection kit (GE Healthcare). Western blot images were quantified using the Multi Gauge version 2.2 software (Fuji Photofilm, Tokyo, Japan).
8
Evaluation of CLA and KPEVs-CLA on H. pylori-induced NF-κB Activation
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AGS cells (1 × 106 cells/well) were seeded in a 6-well plate and incubated for 24 h in complete RPMI without antibiotics. Cells were induced with 3-day-old H. pylori (MOI = 1:100), then CLA or KPEVs-CLA at 0.06 µg/mL prepared in RPMI were added and further incubated for 1 h. RPMI and KPEVs alone were used as untreated and vehicle controls. AGS cells were lysed, and total protein was determined using the Bradford assay. Each sample was subjected to 5%/10% SDS-PAGE and transferred to a PVDF membrane at 100 V for 70 min. The membrane was blocked with 5% BSA/TBS-T for one hour and incubated overnight at 4°C with the following primary antibodies: p-NF-κB p65, NF-κB p65, and GAPDH (Cell signaling technology, USA). The membrane was washed thrice with 1x TBS-T, for ten minutes. HRP-conjugated anti-mouse secondary antibody was added, incubated for 60 min, and washed with 1x TBS-T. Protein signals were developed using the Amersham ECL prime Western blotting detection kit (GE Healthcare, USA). Protein bands were captured using Amersham ImageQuant 800 (Cytiva, MA, USA) and quantified using ImageJ software.
9
Western Blot Analysis of Oct-3/4 in NCCIT Cells
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Untransfected NCCIT cells were lysed with 1 mL of lysis buffer (1% Triton X-100, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HC1, pH = 7.4, 150 mM NaCl, and 0.1% sodium dodecyl sulfate (SDS)), 1% protease inhibitor cocktail, and phosphatase inhibitor (Sigma). Protein concentrations were measured by the Bradford Protein Assay reagent. Briefly, 20 µg of cell lysates were isolated on 12.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). Blocking was carried out with 5% skim milk in Tris-buffered saline (TBS), containing 0.05% Tween 20 (TBS-T). The membrane was then incubated for 1 h with primary mouse anti-Oct-3/4 sc-5279 antibody (Santa Cruz Biotechnology) diluted in 3% blocking buffer and washed with phosphate-buffered saline (PBS) for 30 min at room temperature. A secondary horseradish peroxidase (HRP)-conjugated sheep-anti-mouse antibody (Avicenna Research Institute) was added next and incubated at RT for 1 h. After washing, signals were detected using the Amersham ECL Prime Western Blotting Detection kit (GE Healthcare Life Sciences) and quantified by Gel Logic 2200 (Kodak).
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