The PROTAC-treated MCF7 (human breast cancer) and A549 (human lung cancer) cells were lysed with 1X lysis buffer containing protease inhibitor. Extracted protein lysate (20 μg) were loaded on 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (ATTO, Tokyo, Japan). The membrane was blocked with 3% Bovine Serum Albumin (BSA) in tris-phosphate buffer containing 0.1% Twin 20, and incubated with primary antibodies for 1 h at room temperature. The following antibodies were IGF1Rβ (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Src (1:000, Cell Signaling Technology, Beverly, MA, USA) and GAPDH (1:200, Santa Cruz Biotechnology) as internal control. The signal was reacted with ECL solution (GenDEPOT, Barker, TX, USA) and detected on an Image Quant LAS 4000 biomolecular imager (GE Healthcare, Chicago, Il, USA).
Ecl solution
Manufactured by GenDEPOT
Sourced in United States
ECL solution is a chemiluminescent substrate used for the detection and quantification of proteins in Western blotting applications. It is a reagent used to generate a luminescent signal when interacting with the horseradish peroxidase (HRP) enzyme conjugated to a secondary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (4 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Cellytic buffer, by Merck Group (2 mentions)
N cadherin, by BD (2 mentions)
β actin, by Bioworld Technology (2 mentions)
Snail, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Igf1rβ, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Protease inhibitor, by GenDEPOT (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by BD (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
E cadherin, by BD (1 mentions)
Cytochrome c, by BD (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
12 protocols using ecl solution
1
Analyzing Protein Expression in Cancer Cells
2
Western Blot Analysis of Proteins
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Cell lysate was obtained using 1X RIPA buffer containing a protease inhibitor (GenDEPOT, Barker, TX, USA) and centrifuged at 2000× g rpm for 10 min at 4 °C. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). After blocking with 5% bovine serum albumin for 30 min at 25 °C, the membranes were incubated with each primary antibody according to the manufacturer’s instructions and subsequently incubated with an appropriate secondary antibody (GenDEPOT). The membranes were reacted with ECL solution (GenDEPOT), and protein bands were visualized using X-ray film (CP1000; AGFA, Greenville, SC, USA) or ImageQuant LAS 4000 (GE Healthcare, Piscataway, NJ, USA).
3
Western Blotting Analysis of Apoptosis Markers
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The expression levels of Bcl proteins, cytochrome c, caspases, and PARP were assessed by Western blotting. Briefly, PC-3 cells were seeded at 1×106 per well, incubated at 37°C in 5% CO2 for 18 h, and with various concentrations of FFH for 24 h. Attached cells were then washed twice with ice-cold PBS and total proteins were isolated using protein extraction solution (pro-prep, iNtRON, Gyeonggi-do, Korea). Cell lysates were obtained by centrifugation at 13,250g for 10 min at 4°C. Proteins were separated using sodium-dodecyl sulfate polyacrylamide gel, transferred to PVDF membranes (Millipore, Darmstadt, Germany), which were then blocked by 5% skim milk in TBST buffer for 1 h at room temperature and then incubated for 4°C overnight with specific antibodies for Bcl-2 (1:500), Bax (1:500), cytochrome c (1:200), caspase-9 (1:200), caspase-3 (1:200), PARP (1:500), and β-actin (1:500). After this overnight incubation, horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG, pAb (1:5000), and HRP-conjugated goat anti-mouse IgG pAb (1:3000) were added for 2 h. Membranes were then treated with ECL solution (Gen DEPOT, Houston, TX, USA) and protein expression levels were determined using a photosensitive luminescent analyzer system (Amersham™ Imager 600, UK) and the Image J program (NIH, MA, USA). Values are presented as ratios of densities of respective protein bands to β-actin.
4
Western Blot Analysis of Macrophages
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After removing the culture media of macrophage RAW 264.7 cells, the cells were scraped with CelLytic buffer (Sigma-Aldrich, MO, USA). The cell lysate was centrifuged at 13,000 rpm at 4 °C for 15 min. The protein concentration of the cell lysate supernatant was quantified by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA), and all samples were diluted with CelLytic buffer to have the same concentration of protein (20 μg/20 μL). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-gel was transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA) and blocked with 5% skim milk for 30 min. After blocking, the membrane was incubated with specific primary antibodies at 4 °C overnight. Blots were washed three times with Tris-buffered saline with 0.5% Tween 20 (TBS-T) and then incubated with corresponding horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G for 1 h. ECL solution (GenDEPOT, Barker, TX, USA) was dispensed on the membrane, and protein bands were measured using LuminoGraph (Atto, Tokyo, Japan). Band images and intensities were quantified in the ImageJ program (NIH; Bethesda, MD, USA) and corrected by β-actin levels.
5
Protein Expression Analysis in HCH Cells
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HCH cells were lysed in CelLytic buffer (Sigma-Aldrich, MO, USA) and the lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. The protein concentrations were determined by a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were separated via SDS-PAGE on 10% gels and transferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, and then incubated with specific primary antibodies against β-actin, COX-2, mPGES-1, PGE2, 5-LOX, LTB4, IL-1β, TNFα, IL-6, MMP-2, MMP-3, MMP-9, MMP-13, Bax, Bcl-2, cleaved caspase-9 and -3, cleaved PARP, phospho-NF-κB p65, NF-κB p65, phospho-p38 MAPK and p38 MAPK (dilution 1:1000) overnight at 4 °C. Following primary antibody incubation, the membranes were incubated with the corresponding HRP-conjugated anti-rabbit and anti-mouse IgG secondary antibodies (dilution 1:10,000) for 1 h at 23 °C. The bands were visualized with ECL solution (GenDEPOT, Barker, TX, USA) and the intensity of bands was detected using a LuminoGraph chemiluminescent imaging system (Atto, Tokyo, Japan). β-actin was used as a control for normalization. western blot bands were analyzed using Image J software.
6
Western Blot Analysis of EMT Markers
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Transfected cells with siN-BLRs or siCT were lysed in 1X RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease inhibitor. Isolated proteins were loaded onto 8% to 15% SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Piscat-away, NJ, USA). The membrane was blocked for 1 hour at room temperature in tris–phosphate buffer containing 0.1% Tween 20 with 5% BSA (BD biosciences), before being incubated with primary antibodies at 4°C overnight. The following primary antibodies were used for Western blot analysis: epithelial marker E-cadherin (1:1,000; BD Biosciences), mesenchymal marker N-cadherin (1:1,000; BD Biosciences), vimentin (1:200; Santa Cruz Biotechnology, Dallas, TX, USA, sc-373717), ZEB1 (1:1,000; Cell Signaling Technology), Snail (1:1,000; Cell Signaling Technology), PARP (1:1,000; Cell Signaling Technology, #9542), bcl-xl (1:1,000; Cell Signaling Technology, #2764), Bax (1:1,000; Santa Cruz Biotechnology, sc-493), cytochrome c (1:1,000; BD Biosciences), and β-actin (1:5,000; Bioworld Technology, St. Louis Park, MN, USA, AP0060). The signal was developed in ECL solution (GenDEPOT, Barker, TX, USA) and exposed to an Image Quant LAS 4000 bio-molecular imager for time intervals between 10 seconds and 6 minutes.
7
Protein Expression Quantification by Western Blot
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The expression levels of iNOS and COX-2 were assessed by western blotting. Briefly, RAW 264.7 cells (1χ106 per well) were incubated for 18 hours at 37°C in 5% CO2, and then with various concentrations of FFE for 24 hours. Cells were then stimulated with 50 ng/ml of LPS for 23 hours when attached cells were washed twice with ice-cold PBS. Total proteins were isolated using protein extraction solution (pro-prep, iNtRON, Gyeonggi-do, Korea). Cell lysates were obtained by centrifugation at 4°C at 13,250×g for 10 minutes. Proteins were separated in sodium-dodecyl sulfate polyacrylamide gels, and transferred to PVDF membranes (Millipore, Darmstadt, Germany), which were blocked using 5% skim milk in TBST buffer for 1 hour at room temperature and then incubated overnight at 4°C with specific antibodies of iNOS (1:500), COX-2 (1:1000) and β-actin (1:500). After overnight incubation, Horse Radish Peroxidase (HRP) conjugated Goat anti-rabbit IgG, pAb (1:5000) and Horse Radish Peroxidase (HRP) conjugated Goat anti-mouse IgG pAb (1:3000) were added for 2 hours. Membranes were then treated with ECL solution (GenDEPOT, Houston, TX, USA) and protein bands were detected by a photosensitive luminescent analyzer system (Amersham Imager 600, UK). Protein relative quantities were analyzed using the Image J program (NIH, Maryland, USA) versus β-actin.
8
Investigating MALAT1 Knockdown on Epithelial-Mesenchymal Markers
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MKN74 and AGS cells were transfected with 100 nM siMALAT1 or siCT. The cells were lysed in lysis buffer. Cell lysates were resolved by 8–10% SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Piscataway, NJ, USA). After transfer, membranes were incubated with primary antibodies in 5% BSA (Affymetrix, Inc. Santa Clara, CA, USA) for 24 h at 4 °C. The primary antibodies used were: mesenchymal marker N-cadherin (1:1000, BD Biosciences), snail (1:1000, Cell Signaling Technology), c-Myc (1:500, Santa Cruz), GSK-3β (1:200, Santa Cruz), β-actin (1:5000, Bioworld Technology, Louis Park, MN, USA) and β-catenin (1:500, Santa Cruz). To visualize the target proteins, probed blots were incubated in ECL solution (GenDEPOT, Barker, TX, USA) and exposed to an Image Quant LAS 4000 bio-molecular imager for 10 s to 5 min.
9
Measuring Secreted Hsp90 Proteins
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Cells were lysed in a 1% Triton-X (Cat#H5141) (Biocompare) and protease inhibitor (Cat# ab65621) (Abcam) mixture. Protein concentrations were determined using the Bradford Assay Kit (Bio-Rad). Proteins (20 µg) were separated on 7.5% SDS-PAGE and transferred to PVDF membranes (Cat#162177) (Bio-rad). Membranes were blocked with 5% BSA in TBST, incubated with primary antibodies overnight at 4 °C, then with secondary antibodies for 30 min. Detection was performed using ECL solution (Cat#W3652-020) (GenDEPOT) and a ChemiDoc MP system (Bio-Rad).
For the detection of secreted Hsp90 proteins, the culture medium was collected after ATRA treatment and then concentrated using an Amicon Ultra-15 centrifugal filter unit (Sigma-Aldrich #UFC901008). The concentrated proteins were subsequently analyzed by Western blot.
For the detection of secreted Hsp90 proteins, the culture medium was collected after ATRA treatment and then concentrated using an Amicon Ultra-15 centrifugal filter unit (Sigma-Aldrich #UFC901008). The concentrated proteins were subsequently analyzed by Western blot.
10
Immunoblotting and Immunoprecipitation of HEK293T Cells
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HEK293T cells were co-transfected with the various plasmids and washed with PBS. For immunoblotting, the cells were lysed with 1 × RIPA buffer (GenDEPOT, Barker, TX, USA) containing a protease inhibitor cocktail (GenDEPOT). After sample buffer (100 mM Tris–HCl pH 6.8, 25% glycerol, 5% β-mercaptoethanol, 2% SDS, and 0.1% bromophenol blue) was added to whole cell lysates (WCLs), the mixture was subsequently heated at 94 °C for 5 min. Quantitative proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK), which were blocked with 5% skim milk in Tris-buffered saline (TBS) with 0.1% Tween-20. The indicated antibodies were incubated, and ECL solution (GenDEPOT) was used for detection. To detect HMGB1 secretion, cell culture supernatants were harvested after treatment, and a methanol and chloroform mixture at a 4:1 ratio was added to supernatants to precipitate proteins, and then immunoblotted. For IP, pre-cleared protein G magnetic beads (Bio-Rad, Hercules, CA, USA) were reacted with the indicated antibodies for 1 h at room temperature (RT), and WCLs were incubated with beads overnight at 4 °C. After the beads were washed four times with PBST buffer (0.001% Tween-20 in PBS), the immunoprecipitates were eluted with the sample buffer and separated by SDS-PAGE.
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