Whole cell lysates were prepared using EBC lysis buffer (50 mM Tris-HCl [pH 8.0], 120 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.3 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 0.5% NP40, and 5 U/mL aprotinin) and were then centrifuged. The resulting supernatant (20 µg) was separated on 8% to 12% SDS-PAGE and transferred to PVDF membranes (Invitrogen). The membranes were blocked using 5% skim milk-PBS-0.1% Tween 20 for one hour at room temperature before being incubated overnight with primary antibodies specific for p-CK2α which was purchased from Sigma (St Louis, MO, USA) and CK2α which was purchased from Abcam (Cambridge, UK). p-EGFR (Tyr1173), EGFR, Caspase-3, Akt, p-Erk, Erk, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). P-Akt (Ser473), cleaved PARP (Asp214), Atg7 and LC3 were purchased from Cell Signaling Technology (Berverly, MA, USA). Horseradish peroxidase–conjugated antibodies were used as secondary antibodies. Membranes were developed using ECL kits (PerkinElmer, Waltham, MA, USA).
Ecl kit
Manufactured by PerkinElmer
Sourced in United States
The ECL kit is a laboratory equipment product that enables chemiluminescent detection of proteins in Western blot analysis. It provides reagents for the visualization of target proteins through a light-emitting chemical reaction.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Image lab 3, by Bio-Rad (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Myecl imager, by Thermo Fisher Scientific (3 mentions)
Ab64193, by Abcam (3 mentions)
Hyperfilm ecl, by GE Healthcare (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Anti flag, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Polyscreen, by PerkinElmer (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
75 protocols using ecl kit
1
Whole Cell Lysate Western Blot Analysis
2
Immunoblotting of Protein Extracts
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Total proteins from HEK293T cells were extracted using lysis buffer (150mM NaCl, 50 mM Tris-HCl, pH7.4, 0.5% Triton X-100) adding protease inhibitor cocktail (#11836153001, Roche). Prepared protein samples were separated by 12% or 15% SDS-PAGE and subsequently transferred onto a 0.2μm PVDF membrane (Pall). The immunoblotting was performed with the following primary antibodies (overnight at 4℃) and secondary antibodies (1h at RT): mouseanti-Flag (M2 monoclonal, #F3165, 1:3000, Sigma), rabbit-anti-GAPDH (monoclonal, #2118, 1:1000, Cell Signaling), rabbit-anti-GFP (polyclonal, 1:2000, #ab290, Abcam), rabbit-anti-copGFP (polyclonal, #PA5-22688, 1:5000, Invitrogen), rabbit-anti-Myc (monoclonal, #71D10, 1:1000, Cell Signaling), anti-mouse and anti-rabbit HRP secondary antibodies (1:10000, #A4416 and #A6154, Sigma). The membranes were visualized by ECL kits (#NEL105001EA, PerkinElmer; #34580, Thermo Scientific).
3
Western Blot Analysis of Proteins
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Proteins was released using lysis buffer with protease inhibitors, and were then loaded onto sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels for separation. Next, the proteins were transferred onto a nitrocellulose membrane, which was successively blocked with 5% nonfat milk. Incubation with primary antibodies (Abcam) against the target proteins overnight at 4 °C, followed by washing and incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch). The chemiluminescence signal was examined using ECL kit (PerkinElmer).
4
Quantifying m6A RNA Modifications
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Total RNAs extracted were heated at 98 °C for 3 min. After being chilled on ice immediately thereafter, RNAs were dropped onto a nitrocellulose membrane, followed by crosslink under ultraviolet. The membrane was incubated with blocking buffer (PBS, 0.02% Tween-20, 5% milk, pH7.4) for 1 h at room temperature. Successively, anti-m6A antibody was incubated with the membrane overnight at 4 °C. After washing with PBST, the membrane was incubated with the HRP-conjugated secondary antibody. The signals were examined using ECL kit (PerkinElmer).
5
Protein Extraction and Western Blot Analysis
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Total protein from A549 cells was extracted on ice by the addition of lysis buffer (Beyotime, China) containing protease inhibitors (Sigma, USA). Twenty micrograms of protein was heat denatured in sodium dodecyl sulfate loading buffer (Solarbio, Beijing, China), separated by 10% SDS–polyacrylamide gel electrophoresis, and transferred to PVDF membranes (0.2 μm, Bio-Rad, USA). Membranes were incubated overnight with the indicated primary antibodies, followed by incubation with HRP-conjugated secondary antibodies (Multi-science, Hangzhou, China). Protein bands were visualized by an ECL kit (Perkin Elmer Life Sciences). The original images for western blot are shown in Additional file 1 .
6
Apoptotic Pathway Modulation by CH-AuNP in Gastric Cancer
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The expression of the apoptotic protein after the exposure of different concentrations of CH-AuNP on gastric carcinoma AGS cells was assessed by immunoblotting analysis. The control and CH-AuNP-treated cells were washed with PBS and lysed by sonicating using 200 µL of ice-cold lysis buffer. The sonicated cells were then subjected to centrifugation for 30 minutes at 4°C, 10,000 × g. The supernatant was collected and quantified for protein using Bradford reagent. Fifty micrograms of protein from each group were mixed with sample buffer, boiled for 5 minutes at the temperature of 95°C, and subjected to electrophoresis with 10% SDS-PAGE. The electrophoresed sample was then transferred to a polyvinylidene difluoride membrane blocked with 5% skimmed milk for 1 hour and the membrane was incubated with mouse monoclonal anti-human Bcl-2 (sc-7382), Bcl-xl (sc-8392), Bax (sc-7480), caspase-3 (sc-7272), caspase-9 (sc-56073), and β-actin (sc-47778) antibodies for 12 hours. The secondary anti-mouse horseradish peroxidase-labeled antibodies were added to the membrane after a wash with PBS. The bands of the protein were then viewed using ECL kit, Perkin Elmer, USA.
7
Macrophage Protein Expression Analysis
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Macrophages were seeded at 6.106cells/20cm2-non coated Petri dish and maintained for 24h in serum-containing medium. After phenotype induction (see above), proteins of cell lysates were resolved by SDS-PAGE (4–15% acrylamide, Bio-Rad) and electro-blotted onto PVDF membranes (NEN). After saturation for at least 2h in blotting buffer (50mM Tris, pH 8.0, 90mM NaCl, 2mM CaCl2, 0.15% [w/v] Tween 20 and 5% [w/v] milk powder), membranes were incubated overnight at 4°C with rabbit polyclonal primary antibodies (AQP1 [Chemicon International], total p38 and p38-pT180/Y182 [all from Cell Signaling]) or mouse IgG1 anti-iNOS (BD Transduction Laboratories) or anti-IκBα (Santa Cruz Biotechnology Inc) and anti-pIκBα (Cell Signaling). After 5 washes, blots were incubated at room temperature with the appropriate HRP-conjugated secondary antibodies in 2% BSA-containing buffer for 1h, washed again 5 times, transferred to PBS and revealed by chemoluminescence (ECL kit, Perkin Elmer) using Kodak X-omat blue films and scanned at high resolution (Agfa SNAPscan 600).
8
Western Blot Analysis of Phosphorylated ERK and c-Raf
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The ventricle lysate was prepared in 50 mM Tris HCl [pH 7.5], 150 mM NaCl, 10 mM EDTA, 0.25% Triton X-100, 0.1% NP-40, 1 mM PMSF and cocktail inhibitors (Roche). Protein concentration was measured by the BCA technique. Proteins were separated (100 μg per lane) on an SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. Membranes were blocked for 1 hour at room temperature in 5% non-fat milk with 0.1% Tween 20 in Tris Buffer Saline (pH 7.4), and were probed overnight at 4°C with antibodies: rabbit anti-rat phospho-ERK1/2 (Thr202/Tyr204), mouse anti-rat total ERK1/2 (R & D biosystems; 1:1000), rabbit anti-rat phospho-c-Raf (Ser259) and rabbit anti-rat total c-Raf (Cell signaling; 1:1000). Blots were washed in TBS/0.1%-Tween-20 for 1 hour at room temperature, and incubated with the appropriate HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories; 1/2000) in TBS/0.1% Tween-20/5% nonfat milk for 1 h. Bound antibodies were visualized using enhanced chemiluminescence ECL kit (Perkin-Elmer, Boston, MA) according to the manufacturer’s instructions. Evaluation of the expression of specific proteins was performed with the ImageJ 1.30v Software system.
9
HPV16 L1 Protein Immunoblot Detection
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Equal volume aliquots from organotypic tissue extracts were boiled for 10 minutes in 6% 2 mercaptoethanol (2-ME) loading buffer and loaded onto a 7.5% polyacrylamide gel. Nitrocellulose membranes were blocked using StartingBlock blocking buffer in PBST (Thermo Scientific). To detect HPV16 L1, the anti-HPV16 L1 monoclonal antibody Camvir-1 (BD Pharmigen) was utilized at a 1∶2,000 dilution. An HRP-linked sheep anti-mouse secondary antibody was utilized at a 1∶8,000 dilution. Membranes were washed with PBST after the addition of each antibody. All antibodies were diluted in StartingBlock (Thermo Scientific). HRP was detected using an ECL kit (Perkin Elmer). The conditions for non-reducing L1 immunoblots have been previously described [29] (link). Results are representative of at least 2 different batches of virus preps for each virus type.
10
Western Blot Analysis of Signaling Proteins
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Western blot analysis was conducted using the following primary antibodies: anti-p-Akt (1:5000; ab81283), anti-Cyclin D1 (1:1000; ab226977), anti-p-PI3K (1:1000; ab278545), and anti-beta-actin (1:1000; ab8227). Firstly, VIIb -treated and untreated cells were rinsed with PBS, and then cold lysis buffer was added to induce cell lysis. After centrifugation, the supernatants were collected, and harvested proteins were quantified by Bradford assay and then resolved on SDS-PAGE followed by electroblotting onto polyvinylidene fluoride membranes. Later, membrane blocking was conducted using 5% skimmed milk in 0.1% Tween-20 in PBS (PBST). Then membranes were incubated with the aforementioned primary antibodies at 4 °C. Following 12-h incubation, the membranes were soaked in PBST thrice followed by 1-h incubation with appropriate secondary antibodies. Antibodies were supplied from Abcam (Cambridge, UK). The signals were visualized with a chemiluminescence ECL kit (Perkin Elmer, Waltham, MA, USA) following the manufacturer’s instructions, and images were obtained using a Biorad Imager.
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