Whole-cell lysates were prepared using RIPA buffer with protease inhibitors. After protein quantification (Pierce BCA Protein Assay Kit, Thermo scientific, USA), 20 μg of lysate was separated by electrophoresis on SDS-PAGE gels and blotted onto a polyvinylidene fluoride (PVDF) membrane (Cat#: ISEQ00010, merck milipore). Membranes were incubated with antibodies against EZH2 (Cat#: 5246S, Cell Signaling Technology), Arg1 (Cat#: 93668T, Cell Signaling Technology), iNOS (Cat#: MAB9502-SP, R&D system), or GADPH (Cat#: C1312, Applygen) overnight at 4°C. Then, the membranes were incubated with anti-rabbit/mouse IgG and HRP-linked antibody (Cat#: ZB-2305, ZSGB-Bio) for 1 h at room temperature. After washing twice for 10 min with TBST, the membranes were exposed to obtain the blot images with ECL Reagent (Cat#: P1030-100, Applygen).
Ecl reagent
Manufactured by Applygen
Sourced in China
ECL reagent is a chemiluminescent detection system commonly used in western blotting applications. It functions by producing a luminescent signal upon interaction with the target protein, allowing for the visualization and quantification of protein expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Specific primary antibody, by Abcam (1 mentions)
Goat anti rabbit igg h l secondary antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ecl kit, by Applygen (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Ab137321, by Abcam (1 mentions)
Ab128997, by Abcam (1 mentions)
Ab18203, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Boster Bio (1 mentions)
Super rx n c, by Fujifilm (1 mentions)
17 protocols using ecl reagent
1
Immunoblot Analysis of Protein Expression
2
Protein Expression Analysis of Colon Tissue
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The total protein of colon tissue was extracted using RIPA lysis buffer with the protease inhibitor cocktail (Beyotime Biotechnology, China), and protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology) according to the manufacturer’s instructions. Equivalent amounts of protein (20 μg) were separated by SDS-PAGE and then transferred onto 0.45 μm PVDF membranes (Millipore, Billeria, MA, United States). The membranes were then blocked in 5% nonfat milk in TBST buffer for 1 h at room temperature, followed by incubation with primary antibodies at 4°C overnight. Then membranes were washed with TBST and incubated with secondary antibody for 1 h at room temperature, proteins were detected using ECL reagent (Applygen, China). The antibodies used were as follows: EGFR (1:2,000), PI3K (1:1,000), p-AKT (1:1,000), and β-actin (1:500).
3
Protein Expression Analysis in A549 Cells and Tumor Tissues
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Using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), total proteins were extracted from A549 cells and tumor tissues. Protein concentration was determined by a BCA kit (Beyotime Institute of Biotechnology). The proteins (25 µg) were separated in 10% SDS-PAGE gels and then transferred to PVDF membranes, which were blocked with 5% non-fat dry milk for 1 h at room temperature. After incubation with specific primary antibodies (all purchased from Abcam) at 4°C overnight, the membranes were washed thrice with 1X 0.05% TBST for 10 min and incubated with goat-anti-rabbit IgG H&L secondary antibodies (1:2,000; cat no. ab6721; Abcam) at room temperature for 1 h. Protein bands were visualized by an ECL reagent (Applygen Technologies, Inc.). The primary antibodies were as follows: NPM1 (1:400; cat no. ab183340), phosphorylated (p)-EGFR (1:1,000; cat no. ab40815), EGFR (1:5,000; cat no. ab52894), p-MEK (1:2,500; cat no. ab96379), MEK (1:2,500; cat no. ab32091), p-ERK (1:1,000; cat no. ab201015), ERK (1:10,000; cat no. ab184699), GAPDH (1:2,500; cat no. ab9485). The protein bands were visualized using an ECL kit (Applygen Technologies, Inc.), and the relative protein levels were quantified using ImageJ software 1.8.0 (National Institutes of Health).
4
Protein Isolation and Western Blot Analysis
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Proteins were isolated using a RIPA (radioimmunoprecipitation) lysis buffer. Total protein was separated by 12% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocked with the nonfat milk, the membrane was incubated with antibodies against TWIST (1:1000, Santa Cruz, USA), GAPDH (1:5000, Santa Cruz, USA). The membrane was measured by ECL reagent (Applygen, Beijing).
5
Protein Expression Analysis Protocol
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Cells were lysed with RIPA buffer (Beyotime) containing protease inhibitors (04693132001, complete EDTA-free, Roche, Basel, Switzerland), and the supernatant was collected by centrifugation at 4 °C. The concentrations of proteins were quantified using the BCA assay kit (P0010, Beyotime, Shanghai, China), and 30 μg of protein from each sample was separated on a 12% SDS-PAGE gel. The proteins were transferred to a PVDF membrane, which was blocked in 5% bovine serum albumin (BSA) for 2 h at room temperature. The PVDF membrane was incubated with the primary antibodies (a list of specific antibodies is provided in Table S2 ) overnight at 4 °C. Secondary antibody conjugated to horseradish peroxidase was incubated with the PVDF membrane for 1 h at room temperature, and target proteins were detected by enhanced chemiluminescence (ECL) reagent (Applygen, Beijing, China). The bands were imaged by an imaging system (Sagecreation, Beijing, China), and quantified with Quantity One software (Bio-Rad, Hercules, CA, USA).
6
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer was used to extract the total proteins from treated glioma cells, and the protein concentration in each extract was quantified using a BCA protein assay kit (Amresco, Fountain Parkway Solon, OH, USA, FA016-50G). An aliquot of total protein from each treatment group was separated by 10% SDS-PAGE, and the protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA, IPVH00010). The membranes were subsequently blocked with non-fat milk and then incubated with primary antibodies against RAB3D (1:10,000, Abcam, Cambridge, UK, ab128997), E-cadherin (1:1,000; Abcam, ab76055), N-cadherin (1:500; Abcam, ab18203), vimentin (1:500; Abcam, ab137321) or GAPDH (1:2,000; Abcam, ab8245) for 1 h at room temperature. After washing, the membranes were incubated with an HRP-conjugated secondary antibody (1:20,000; BOSTER, Pleasanton, CA, USA, BA1054) for 40 min. The immunostained proteins were visualized by using ECL reagent (Applygen Technologies, Beijing, China) and X-ray film (SUPER RX-N-C; Fuji, Japan).
7
Quantification of Phospho-JAK2 and Phospho-STAT3
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The liver tissues were homogenized with lysis buffer (Applygen, China) according to the manufacturer’s protocol. The protein extracts were separated by 8% SDS-PAGE electrophoresis and electro-transferred to PVDF membranes (Millipore, MA, United States). After blocking with 5% BSA, the membranes were incubated overnight at 4°C with specific primary antibodies against Phospho-JAK2 (Cell Signaling Technology, Danvers, MA, United States), Phospho-STAT3 (Cell Signaling Technology, Danvers, MA, United States), and β-actin (Cell Signaling Technology, Danvers, MA, United States) separately. Following four washes with Tris-buffered saline tween-20 (TBST), the membranes were incubated with the corresponding secondary antibodies as Goat Anti-Rabbit IgG (Lablead, China) and for 1 h at room temperature. Membranes were then washed four times with TBST, and the detection was carried out with an electrochemiluminescence (ECL) reagent (Applygen, China). The blot was imaged using EvolutionCapt FX6 and subjected to quantification analysis using Image J software. The results are expressed as the ratio of the relative intensity of the target proteins to that of the internal standard. JAK2 and STAT3 blots were performed by stripping and re-probing the blots with corresponding antibodies, respectively (Cell Signaling Technology, Danvers, MA, United States).
8
Immunoblotting Procedure for Protein Expression
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After 48 h of transfection in six-well plates,the total protein from cells was extracted using RIPA (Beyotime Biotechnology,China).The BCA method was used to detect the protein concentration. The same amounts of protein samples (40 µg) were separated by 12% SDS-PAGE, and then transferred to polyvinylidene uoride (PVDF) membranes. Next, the membranes were blocked with 5% skim milk powder at room temperature for 2h. The membranes were washed three times with TBST for 10 min each time. Next,the membranes were incubated with primary antibodies overnight at 4℃. The primary antibodies used were as follows:PCMT1(10519-1-AP,Proteintech), AKT(Ab8805,Abcam), P-AKT(Thr 308, Ab38449, Abcam), GSK-3β (AF5016, A nity), P-GSK-3β(Ser 9,AF2016,A nity), Snail (AF6032,A nity), E-cadherin (Ab76055,Abcam),N-cadherin(AF5239,A nity), caspase-3 (Ab184787,Abcam), Bax (AF0120,A nity), and Bcl2 (AF6139,A nity). The membranes were washed twice as described above and incubated with homologous secondary antibodies (BOSTER, China) at room temperature for 2 h.The membranes were washed as described above. ECL reagent (Applygen, China) was added to the membranes and the membranes were placed on an automatic chemiluminescence imaging device (Bio-Rad,USA) to develop.
ImageJ software was used to perform grayscale analysis.
ImageJ software was used to perform grayscale analysis.
9
Western Blot Analysis of Protein Expression
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Differential protein expression between the two cell groups was confirmed by Western blotting and further verified using independent samples. SiRNA-transfected cells were harvested at 72 hours after transfection. Whole-cell proteins were extracted as described above and subjected to 12% SDS-PAGE followed by transfer onto a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated with primary antibodies (b-actin: 1:1000 [Santa Cruz Biotechnology, Dallas, TX, USA], SPARC: 1:1000 [Cell Signaling Technology, Danvers, MA, USA], PTCH1: 1:1000 [Abcam, Cambridge, UK]) overnight with shaking at 4°C. Membranes were then washed three times with Tris buffered saline-Tween 20 for 5 minutes. Then, secondary anti-mouse or anti-rabbit (Cell Signaling Technology) antibodies were added and incubated at room temperature for 1 hour. After washing, immunoreactive proteins were detected using an enhanced chemiluminescence (ECL) reagent (Applygen Technology, Beijing, China).
10
Western Blot Analysis of EMT Markers
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50 μg of protein samples from lysed cells went through 10% SDS-PAGE before transfer to nitrocellulose membranes. The membranes were blocked with skim milk for 2 h. Thereafter, the primary antibodies against SOX4 (1:1,000, catalog no. ab80261, Abcam, UK), E-cadherin (1:1,000, catalog no. ab76055, Abcam, UK), vimentin (1:1,000, catalog no. ab925475, Abcam, UK), fibronectin (1:1,000, catalog no. ab24135, Abcam, UK), CCT4 (1:1,000, catalog no. 21524-1-AP, Proteintech, IL, USA), and GAPDH (1:1,000, catalog no. ab1816025, Abcam, UK) were incubated with the membranes overnight, and horseradish peroxidase (HRP)-conjugated secondary antibodies were used for subsequent incubation. Immunocomplex visualization was enhanced using enhanced chemiluminescence (ECL) reagent (Applygen, Beijing, China).
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