The transfection-mediated co-immunoprecipitation (Co-IP) assay was employed to identify the physical interaction of EBNA2 with BS69CC-MYND using EBV-negative B lymphoma cells (BJAB). Cells were co-transfected with the expression plasmids of EBNA2 (E2) and flag- BS69CC-MYND wild type, Q546A mutant (Q546A), or Q546A/W550A mutant (Q546A/W550A) and subjected to IP analysis using M2-conjugated sepharose (Sigma). The immunoprecipitated samples were subjected to SDS-PAGE, followed by Western blotting with antibodies for EBNA2 (Millipore) and M2 (Sigma), respectively. The antibody for actin internal control was purchased from Santa Cruz. The proteins were detected and visualized using an ECL detection kit (Millipore). ChIP assays were performed using BJAB cells that have been transfected with LMP1-promoter reporter plasmid (LMP1-Luc) and flag-BS69CC-MYND wild type or Q546A, with or without E2 using M2-conjugated sepharose and IgG negative control (Millipore) following the previously described protocol [53). The protein levels of endogenous BS69 in the obtained cell lines were determined by Western blot using BS69 antibody (Santa Cruz biotechnology). The antibodies for immune blots are 6F9/60 (Novas Biologicals) for EBNA1, MABE8 (Millipore) for EBNA2, and C4 (Santa cruz) for actin internal control. The proteins were detected and visualized using an ECL detection kit (Millipore).
Ecl detection kit
Manufactured by Merck Group
Sourced in United States, Germany, China, United Kingdom, France, Japan
The ECL detection kit is a lab equipment product developed by Merck Group. It is designed to detect and quantify proteins or other biomolecules through an electrochemiluminescence (ECL) process. The kit includes all the necessary reagents and consumables to perform ECL-based assays in a research or diagnostic laboratory setting.
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Lab products found in correlation
Pvdf membrane, by Merck Group (47 mentions)
Ripa buffer, by Beyotime (18 mentions)
Ripa lysis buffer, by Beyotime (12 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Quantity one software, by Bio-Rad (9 mentions)
Las 4000, by Cytiva (8 mentions)
Bca kit, by Beyotime (7 mentions)
Imagequant las 4000 mini, by GE Healthcare (7 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Protein assay kit, by Bio-Rad (6 mentions)
Bca protein assay kit, by Beyotime (6 mentions)
β actin, by Abcam (5 mentions)
Gapdh, by Cell Signaling Technology (5 mentions)
Nitrocellulose membrane, by Merck Group (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Lysis buffer, by Beyotime (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (4 mentions)
214 protocols using ecl detection kit
1
EBNA2-BS69 Protein Interaction Assay
2
Protein Extraction and Western Blot Analysis
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Cells were harvested and lysed with lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM DTT, 1 mM sodium orthovanadate, 1 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride] on ice for 1 h. Afterwards, cell lysates were centrifuged for 15 min at 11,000 × g at 4°C. Protein concentrations of the supernatants were determined using the bicinchoninic acid assay (BCA) assay. A total of 50 mg of protein loaded per lane and separated using 8–12% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were then blocked with 5% non-fat milk and incubated 4°C for 12 h with the specified primary and secondary antibodies. All antibodies were diluted at 1:1,000 for western blot analysis. Protein bands were visualized using an ECL detection kit (EMD Millipore).
3
Quantitative Western Blot Analysis
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Total proteins of the cells were extracted using protein extraction kit (Beyotime). Concentrations of proteins were determined using BCA kit (Beyotime). Then, the proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferred onto PVDF membranes (Thermo Fisher). Primary antibodies against SRSF1 (1:2000, Abcam, Cambridge, MA, USA), CDK2 (1:1000, Abcam), CyclinE1 (1:1000, Abcam), β-actin (1:1000, Abcam) were incubated with the membranes at 4°C for 12 h. After washing with TBST for three times, the membranes were then incubated with corresponding HRP-conjugated secondary antibodies (1:5000, Abcam) at room temperature for 2 h. The protein samples were visualized using an ECL detection kit (Merck Millipore, Billerica, MA, USA). The integrated density of each band was normalized to the corresponding β-actin band.
4
Western Blotting of Cell Signaling Proteins
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Cells were washed 3 times with PBS and treated with RIPA lysis buffer (#89900, Thermo Fisher, Waltham, MA, USA) mixed with protease and phosphatase inhibitor (Roche, Basel, Switzerland). Ten to twenty micrograms of total protein from each sample was resolved on a 10% PAGE gel and transferred to a polyvinylidene difluoride (PVDF, Merck Millipore, Germany) membrane. The blots were then probed with antibodies against GAPDH (1:10000, KangChen, Shanghai, China), STAT3 (1:1000, #4904, Cell Signaling Technology, USA), pSTAT3 (Tyr705) (1:1000, #4903, Cell Signaling Technology, USA), HIC1 (1:5000, #H8539, Sigma-Aldrich, Saint Louis, MO, USA) and cyclin D1 (1:1000, #2978, Cell Signaling Technology), followed by incubation with peroxidase-labeled secondary antibodies. Immunoreactive proteins were detected by enhanced chemiluminescence (ECL) detection kit (Merck Millipore, Germany).
5
Quantifying Protein Expression in Cells
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To detect proteins in whole cell lysates, cells were washed with ice-cold PBS and lysed using a protein extraction kit (GE Healthcare, Piscataway, NJ, USA). Proteins (30 μg) were separated by 10% SDS-PAGE and bands were stained with Coomassie Blue R-250 (ThermoFisher scientific, MA, USA). Proteins were electrophoretically transferred to polyvinylidene difluoride membranes. The transferred membrane was blocked with 5% skim milk for 1 h and incubated with primary antibodies against PEPCK, G6Pase, AMPKα, phospho-AMPKα (Thr-172), or β-actin. Beta-actin was used as a loading control. The membrane was washed three times with TBST (100 mM Tris pH 7.4, 150 mM NaCl, 0.5% Tween-20) for 10 min and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) secondary antibodies. Signal was detected using a Fujifilm luminescent mage analyzer LAS4000 with ECL detection kit (Merck Millipore, Darmstadt, Germany). Three or four separate experiments were performed with different samples.
6
Western Blot Analysis of Protein Signaling
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Total cell extracts (20 μg of protein) were fractionated by 10–12% SDS–PAGE, transferred to an immobilon polyvinylidene difluoride membrane (Amersham Biosciences, Corston, UK), and probed with one of the specific primary antibodies against PTP4A3 (1:1000, GTX100600, GeneTex, Hsinchu, Taiwan), p-STAT3 (1:1000, GTX104616, GeneTex, Hsinchu, Taiwan), STAT3 (1:1000, GTX11800, GeneTex, Hsinchu, Taiwan), STAT5A (1:1000, GTX113351, GeneTex, Hsinchu, Taiwan), FOXO3A (1:1000, GTX100277, GeneTex, Hsinchu, Taiwan), TERT (1:1500, GTX124242, GeneTex, Hsinchu, Taiwan), TOP2A (1:1500, GTX100689, GeneTex, Hsinchu, Taiwan) p-JAK2 (1:1000, GTX132784, GeneTex, Hsinchu, Taiwan), JAK2 (1:1000, GTX01195, GeneTex, Hsinchu, Taiwan), p-P65 (1:1000, GTX133899, GeneTex, Hsinchu, Taiwan), P65 (1:1000, GTX102090, GeneTex, Hsinchu, Taiwan) and β-actin (1:10,000, #4970, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Subsequently, the membrane was hybridized with appropriate HRP–conjugated secondary antibodies for 1 h at room temperature. Finally, immune complexes were visualized using the chemiluminescence method with an ECL detection kit (Merck Millipore, Temecula, CA, USA) on Fujifilm LAS-3000 (Fujifilm, Tokyo, Japan).
7
Protein Expression and Functional Assays
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Primary antibodies: Flag antibodies (M2, Sigma and MDBio, Taiwan), Actin monoclonal antibody (Millipore, Billerica, MA, USA), GATA1 antibody (MDBio), CLDN11 antibodies (Western/ab53041, Abcam, Cambridge, UK; IHC/SC25711, Santa Cruz, Dallas, TX), Tubulin alpha IB (ab108629, Abcam) and Tubulin beta III antibodies (ab52623, Abcam). Secondary antibodies: HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies (Gene Teks) were used. Western blot results were visualized by ECL detection kit (Immobilon, Merck/Millipore, Germany). Recombinant proteins GST-GATA1 (H00002623, Abnova, Taiwan) and GST-CLDN11 (H00005010, Abnova) were used in EMSA and tubulin polymerization assay, respectively.
8
Western Blot Analysis of IRS and PI3K Signaling
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The expression of proteins was determined by western blot as previously described (18 (link)). The concentration of protein was detected using a BCA kit (Beyotime, Shanghai, China). Then, 30 μg of total protein was loaded onto 8% SDS polyacrylamide gels and transferred to the polyvinylidene fluoride membrane. The membrane then was blocked by 5% BSA (Sigma) for 1 h and incubated with primary antibodies at 4°C overnight. The next day, the membrane was washed with PBS for three times and then followed by incubation with HRP-labeled goat anti-rabbit IgG (Abcam, America) secondary antibody at 37°C for 1 h. Signals were detected by using the ECL detection kit (Merck Millipore) and normalized to the expression of GAPDH. The antibodies included Total insulin receptor substrate (IRS)-1 (ab52167, 1:1,000, Abcam), phosphorylated IRS-1 (p-IRS-1) at PY896 (sc-560, 1:500, Santa Cruz), total IRS-2 (ab52606, 1:1,000, Abcam), phosphorylated IRS-2 (p-IRS-2) at S731 (sc-1555-R,1:500, Santa Cruz), total PI3K (ab154598,1:1,000, Abcam), phosphorylated PI3K (p-PI3K) at P85 (ab191606, 1:1,000, Abcam), phosphorylated Akt (p-Akt) at Ser-473 (ab192623, 1:1,000, Abcam), GAPDH (ab9485, 1:1,000, Abcam). The absorbance of each band was quantitated using the Image Pro Plus Software.
9
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Whole-cell and tissue lysates (pooled tissues from each group) were prepared using RIPA lysis buffer. The concentrations of extracted proteins were calculated using BCA kits (Beyotime). Approximately 50 μg of protein was separated by 8–12% SDS–PAGE and transferred to a PFDV membrane. The membrane was then blocked with 10% skimmed milk for 2 h at 37°C. After blocking, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-E-cadherin (1:1,000), anti-vimentin (1:1,000), anti-β-actin (1:1,0000), anti-cleaved caspase-3 (1:1,000), anti-cleaved PARP (1:1,000), anti-c-myc (1:1,000), anti-Snail (1:1,000), anti-N-cadherin (1:1,000), anti-cyclin D1 (1:1,000) (all from Cell Signaling Technologies, Boston, USA), anti-BAX (1:800), anti-Bcl-2 (1:800) (both from ProteinTech Group, Chicago, IL, USA), and anti-β-catenin (1:5,000; Abcam, Cambridge, UK). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam) at 37°C for 2 h. Finally, the protein bands were detected using an ECL detection kit (Merck, Millipore, USA).
10
Purification and Characterization of pmTSP-II Proteins
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Approximately 10 µg of purified pmTSP-II proteins were dissolved in either native loading buffer or SDS-PAGE loading buffer. Each sample was then electrophoresed on 12.5% polyacrylamide under both non-reducing and reducing conditions. The gels were then stained by FASTsilver™ Gel staining kit (Merck, Darmstadt, Germany) or Coomassie blue staining (Merck, Darmstadt, Germany). For Western blotting, both native and reducing gels were electro- transferred onto a PVDF membrane (Merck-Millipore, Darmstadt, Germany). The membrane was treated with a blocking solution (1% BSA in PBS containing 0.1% tween) for 1 h at room temperature and then incubated with 0.4 μg/ml (1:500) of a CTD110.6 monoclonal antibody overnight at 4 °C. After an extensive wash with PBST, the membrane was further incubated with 1:2,500 horse radish peroxidase (HRP) conjugated goat anti-mouse IgG (Abcam, Cambridge, UK) (room temperature, 2 h). The antigen–antibody complex was visualized by enhanced chemiluminescent method using ECL detection kit (Merck, Darmstadt, Germany). The resolved proteins on the PVDF membrane were exposed to the anti-TSP antibody16 (link) and the corresponding HRP-conjugated secondary antibody in the same conditions described above.
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