Total cellular proteins were extracted, quantified, and subjected to gel electrophoresis according to standard procedures as we described previously [35 (link)]. The antibodies used in this study included anti-HO-1 (ab52947; Abcam, Cambridge, UK), anti-Cyclin D1 (#2978), anti-CDK4 (#12790), anti-p21 Waf1/Cip1 (#2947), anti-p27 Kip1 (#3686), and anti-actin (A5441; Sigma-Aldrich). Antibodies were obtained from Cell Signaling Technology, Danvers, MA, USA, unless specified otherwise. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham ECL Detection System; GE Healthcare, Piscataway, NJ, USA).
Amersham ecl detection system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Amersham ECL detection system is a lab equipment product designed for chemiluminescent detection. It is used to detect and analyze proteins in western blot applications.
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Lab products found in correlation
Anti actin, by Merck Group (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Las 4000 luminescent image analyzer, by GE Healthcare (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Multi gauge software, by Fujifilm (2 mentions)
Modified lowry protein assay, by Thermo Fisher Scientific (2 mentions)
Ab52947, by Abcam (1 mentions)
Anti cyclin d1, by Merck Group (1 mentions)
Anti cdk4, by Merck Group (1 mentions)
Anti p21 waf1 cip1, by Merck Group (1 mentions)
Anti p27 kip1, by Merck Group (1 mentions)
Polyvinylidene membrane, by Merck Group (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti tlr4 antibody, by Novus Biologicals (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Actin, by Cell Signaling Technology (1 mentions)
Phospho p38 mapk, by Cell Signaling Technology (1 mentions)
21 protocols using amersham ecl detection system
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Heme Oxygenase-1
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The tissue samples were washed twice with phosphate buffered saline (PBS), homogenized, and lysed in the RIPA buffer (Upstate, Billerica, MA) for 20 min at 4 °C. Lysates were centrifuged at 12,000 g at 4°C for 12 min and we separated supernatants for western analysis. SDS-PAGE (10% gel) was used to separated equal amount (40 mg) of cell lysates, followed by transferring them to polyvinylidene membranes (Millipore, Billerica, MA), and then probed them with antibodies against proteins of interest, including anti-Heme oxygenase1 antibody (1:1000, room temperature for 2 h, Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin antibody (1:10000, room temperature for 1 h, Santa Cruz Biotechnology, Santa Cruz, CA). Appropriate horse radish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were adopted to incubate blots. Enhanced chemiluminescence (ECK) kit (Amersham ECL detection system, GE Healthcare) was used to visualize bound antibodies, and the relative density of the target bands was determined by using densitometry analysis.
3
Intrarenal TLR-2 and TLR-4 Expression
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Intrarenal Toll-like receptors 2 and 4 (TLR-2 and TLR-4) were analyzed by western blot analysis. According to the manufacturer's instructions, equal amounts of whole kidney protein extract (30 μg) were separated by electrophoresis on a NuPAGE Bolt mini gel system (Thermo Fisher Scientific). The gels were transferred onto a nitrocellulose membrane using an iBlot 2 Dry Blotting System (Thermo Fisher Scientific) after electrophoresis. Membranes were blocked with 5% skim milk tris-buffered saline solution with 0.1% Tween20 (TBST) for 1 h at room temperature and then incubated overnight at 4°C with one of the following antibodies: mouse monoclonal anti-TLR2 antibody (MyBioSource, San Diego, CA) or anti-TLR4 antibody (Novus Biologicals, Centennial, CO). The horseradish peroxidase-conjugated secondary antibody was applied for 30 min at room temperature after washing with TBST. The signal was visualized using an Amersham ECL detection system (GE Healthcare, Chicago, IL), following the manufacturer's instructions. Bands were densitometrically analyzed using ImageJ 1.8 software (Wayne Rasband, National Institutes of Health, MD) and normalized against corresponding β-actin band intensity as an internal control.
4
Western Blot Analysis of α-SMA in Kidney
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Details of Western blot analysis to evaluate α-SMA have been described.35 (link) Briefly, kidney samples were homogenized and equal amounts of protein samples (30 μg) were electrophoresed on Life Technologies NuPAGE® sodium dodecyl sulfate-polyacrylamide gels (Thermo Fisher Scientific), and transferred onto Immobilon™-P membranes (Merck Millipore, Billerica, MA, USA). After blocking, the membranes were incubated overnight at 4°C with appropriate dilutions of primary antibodies against α-SMA (Abcam plc, Cambridge, England), and GAPDH (Santa Cruz Biotechnology Inc, Dallas, TX, USA). After washing, the membrane was incubated for 1 hour at room temperature with a secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin; GE Healthcare UK Ltd, Little Chalfont, UK). Antibody complexes were visualized using the Amersham ECL detection system (GE Healthcare UK Ltd) as directed by the manufacturer. Protein was quantified densitometrically using ImageJ 1.480 software. The expression levels of α-SMA were normalized relative to that of GAPDH. Data are expressed as relative to the sham-operation group.
5
SSTR2 Peptide Modulates Cell Signaling
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After 48 hr treatment of SSTR2 peptide (1 and 10 pM/nM/μM), C6 cells were harvested in lysis buffer and whole cell lysates were prepared using standard protocol. After nanoformulation treatment whole cell lysates were prepared from rat glioma tissues and C6 cells of treated and untreated batches by standard procedures. 70 μg of cellular proteins were separated on SDS-PAGE and subjected to IB using primary antibodies against Phospho-EGFR (Y1068), EGFR, AKT, Phospho-AKT (S473), STAT3, Phospho-STAT3 (Y705), ERK1/2, Phospho-ERK1/2(Y202/T204), Phospho-p38 MAPK, p38 MAPK, p21 and Actin (Cell Signaling Technology and Santa Cruz Biotechnology). Actin was used as loading controls. Bands were detected using the Amersham ECL detection system (GE Healthcare).
6
Protein Expression Analysis by Western Blot
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Total cellular proteins were extracted, quantified, and subjected to gel electrophoresis according to standard procedures as we described previously [49] (link). The antibodies used in our study, including anti-caspase 3 (#9662), anti-cleaved caspase 3 (#9661), anti-caspase 7 (#9494), anti-poly(ADP-ribose) polymerase (PARP) (P248; Sigma), anti-Bax (#2774), anti-Bcl-2 (#2872), anti-phospho-ERK (#9101), anti-ERK (#9102), anti-phospho-p38 (#4511), anti-p38 (#9212), anti-phospho-JNK (#9255), anti-JNK (#9252), and anti-actin (A5441; Sigma). Antibodies were obtained from Cell Signaling, Danvers, MA unless specified otherwise. The antigen-antibody complexes were visualized with by chemiluminescence with the Amersham ECL detection system (GE Healthcare, Piscataway, NJ).
7
Optic Nerve Protein Expression Analysis
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Thirty-six rats (NG: 18 rats; HG: 18 rats) were euthanatized 1 week after intravitreal administrations for immunoblot analysis. Four mm optic nerves from immediately behind the eye ball were homogenized in protein extraction buffer. Since the optic nerve pieces were small, each sample included two optic nerves. Equal amount of proteins (3 µg) determined by the Bradford assay was applied and loaded. Then, samples were transferred to PVDF membranes. After blocking, membranes were exposed with the primary antibodies: Anti-Beclin-1 antibody for overnight (1:200; Medical & Biological Laboratories, Co., Ltd., Nagoya, Japan) or anti-β-actin antibody for 2 h (1:500; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). After three times washing, membranes were reacted with the secondary antibodies: Rabbit IgG or mouse IgG. Immunoblots were evaluated with the Amersham ECL detection system (GE Healthcare Life Sciences, Little Chalfont, UK).
8
Quantifying Protein Levels via Western Blot
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The protein content of tissue or cell extracts was quantified using the Bradford reagent (Bio-Rad). The samples were mixed with 5x Laemmli sample buffer and boiled for 5 min. The boiled samples (15 μg protein) were separated on 10% SDS polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were incubated with a blocking buffer (5% nonfat milk or 1% BSA in Tris-buffered saline containing 0.5% Tween-20) for 1 hr with rocking at room temperature. The blots were incubated with the indicated primary antibodies in the blocking buffer at 4°C overnight. After washing three times with TBST, the blots were incubated with horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin G (IgG; 1:5000) or with goat anti-rabbit IgG (1:3000) for 1 hr at room temperature. The specific immune-reactive protein bands were visualized using Amersham ECL detection system (GE healthcare).
9
Western Blot Analysis of Protein Lysates
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Cell lysates were prepared in lysis buffer (PBS containing 1% (v/v) protease inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA) and 0.5% (v/v) nonidet-P40 (Sigma, Tokyo, Japan). Cell lysates (total protein 10 μg per sample) were resolved on 4–12% NuPAGE Bis-Tris gels (Invitrogen, Waltham, MA, USA) followed by transfer to nitrocellulose membranes (Millipore, Burlington, MA, USA). The membranes were subsequently blocked with TBST (20 mm Tris-HCl pH 7·5, 150 mm NaCl, 0·1% Tween-20) containing 5% (w/v) skimmed milk powder, and probed with various antibodies as indicated in TBST containing 2.5% (w/v) skimmed milk powder. Affinity purified HRP-linked secondary antibodies (Cell Signaling Technology) were used for detection, and the membranes were developed using the Amersham ECL detection system (GE Healthcare, Mississauga, ON, Canada) according to the manufacturer’s instructions.
10
Western Blot Analysis of CDK6 Expression
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PASMCs were lysed with lysis buffer, and the lysate was loaded onto a SDS-PAGE gel (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and then blotted them onto a nitrocellulose membrane (Millipore, Billerica, MA). We blocked the membrane in phosphate-buffered saline with 5% nonfat dry milk and incubated it for 1 h. Subsequently, the membrane was incubated with primary antibody of anti-β-actin (1:1,0000; Sigma-Aldrich) and primary antibody of CDK6 (1:1,000; Cell Signaling Technology, Beverly, MA) overnight at 4°C. HRP-labeled secondary antibody (1:1,0000; Cell Signaling Technology, Beverly, MA) was used to treat the primary antibody-labeled membranes for 1 h. An ECL kit (Amersham ECL detection system, GE Healthcare) was used to detect the signals in the blots. The density of the β-actin band was used to normalize the value of the relative density of the target protein band.
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