For immunoblot analysis, HEK293 T-REx drTPRM7 wild type or truncation mutant cells were induced for 16 h with 1 μg/mL tetracycline added to the media. Cells were harvested and dissolved in lysis buffer (10 mM Tris-HCl, 75 mM NaCl, 5% glycerol, 0.5% triton, 5 mM EDTA, 1 mM PMSF) containing protease inhibitors (104 mM AEBSF, 80 μM Aprotinin, 4 mM Bestatin, 1.4 mM E-64, 2 mM Leupeptin and 1.5 mM Pepstatin A) for 30 minutes at 4 °C. After incubation, the lysates were centrifuged at 12,000 rpm for 5 min. at 4 °C and the protein concentration of the lysates were measured using a protein assay reagent (Thermo Scientific, USA). Soluble lysates were boiled with NuPAGE LDS sample buffer and NuPAGE Reducing Agent (Invitrogen) at 95 °C for 8 minutes. Equal amounts of proteins (40 μg) were loaded and separated in a NuPAGE 4–12% gel (Invitrogen) then transferred to a PVDF membrane. Proteins were detected by immunoblotting with the antibodies anti-HA (3F10, Roche), anti-GAPDH (6C5, Abcam, UK) followed by treatment with horseradish peroxidase (HRP)-conjugated anti-rat or anti-mouse antibody (GE Healthcare, USA). GAPDH was evaluated as an internal control. The antibody-bound protein was visualized using ECL solution (Life Technologies, USA.)
Ecl solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom
ECL (Enhanced Chemiluminescence) solution is a laboratory reagent used in Western blotting techniques. It is designed to detect and visualize proteins immobilized on a membrane by producing a light-emitting chemical reaction. The solution contains the necessary components to enable the chemiluminescent detection of target proteins labeled with enzyme-conjugated antibodies.
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Lab products found in correlation
Pvdf membrane, by Merck Group (17 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Ripa buffer, by Thermo Fisher Scientific (11 mentions)
Bca protein assay kit, by Beyotime (6 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Gapdh, by Abcam (4 mentions)
Chemidoc imaging system, by Bio-Rad (4 mentions)
Bca method, by Beyotime (4 mentions)
Protease inhibitor cocktail, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Chemidoc, by Bio-Rad (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Ripa buffer, by Merck Group (3 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
Cleaved parp, by Cell Signaling Technology (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
151 protocols using ecl solution
1
Immunoblotting of TPRM7 Variants in HEK293 Cells
2
Cerebellar αCAMKII Regulation by Extracellular K+
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Cerebellar tissue slices lysates of 150 μm thickness were incubated for 10 min in any of three conditions: low [K]o (1 mM), high [K]o (50 mM), or high [K]o with mibefradil (1 μM) and Ni2+ (300 μM). Homogenates were then made in the same solutions using a hand held glass homogenizer. Eluted lysates were loaded on 6–10% Tris-glycine gel and resolved using SDS-PAGE. Samples were transferred to 0.2 μm PVDF membrane (Millipore, Etobicoke, ON, Canada) and Western blot analysis performed using a monoclonal mouse anti-αCAMKII (1:1000; Santa Cruz, Dallas, TX, USA) or a polyclonal rabbit anti-p-αCAMKII (Thr286) (1:1000; Santa Cruz, Dallas, TX, USA). The secondary antibodies used were the appropriate mouse or rabbit antibodies conjugated to HRP (1:5000; Molecular Probes, Eugene, OR, USA) and reacted with ECL solution (Life Technologies, Burlington, ON, Canada).
3
Western Blot Analysis of Protein Expression
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After isolating the total proteins from the treated HrGECs, a bicinchoninic acid (BCA) protein assay kit (Merck, New Jersey, USA) was utilized to determine the concentration of proteins and about 40 μg proteins were loaded, followed by being separated with the 12% SDS-PAGE. Then, the proteins in the SDS-PAGE were transferred to the PVDF membrane (Merck, New Jersey, USA) and the membrane was subsequently incubated with 5% BSA for 2 hours, followed by being incubated with the primary antibody against NADPH oxidase-2 (NOX-2) (1:1200, Zymed, California, USA), NLRP3 (1:1500, Zymed, California, USA), apoptosis-associated speck-like protein containing a CARD (ASC) (1:800, Zymed, California, USA), p-AMPKα (1:500, Zymed, California, USA), AMPKα (1:1600, Zymed, California, USA), mTOR (1:1500, Zymed, California, USA), and β-actin (1:8000, Zymed, California, USA) overnight. The membrane was exposed to enhanced chemiluminescence (ECL) solution (Invitrogen, California, USA) after 1.5 hours of incubation in secondary antibody solution (1:2000, Zymed, California, USA) and the relative expression level of target proteins was confirmed by visualization with Image J software [16 (link)].
4
Protein Expression Analysis in MDA-MB-231 Cells
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Total protein was extracted from MDA-MB-231 cells treated with indicated nanocomplexes using a RIPA lysis reagent (Beyotime, China), quantified by using a BCA kit (Beyotime), separated in the SDS-PAGE gel and transferred to PVDF membranes. Subsequently, the blots were soaked in blocking buffer for 15 minutes, and incubated in diluted primary antibodies against Rictor (ab105469), Akt (ab8805), phosphorylated-Akt (ab38449), p70s6k (ab32359), phosphorylated p70s6k (ab59208), and tubulin (ab7291) as internal control, overnight at 4°C. Next day, the blots were washed with PBS and incubated with HRP-conjugated secondary anti-mouse or anti-rabbit antibody, accordingly. All antibodies used in this work were purchased from Abcam (USA) and diluted as instructions of the manufacturer. The blots were visualized by using the ECL solution (Invitrogen) and captured in a gel imaging system (BioRad).
5
Protein Expression Analysis by Western Blot
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Following isolation of the total proteins from the treated cells, a BCA kit (Merck, New Jersey, USA) was used to determine the concentration of proteins, and about 40 μg of protein was loaded, followed by separation with 12% SDS-PAGE. Then, the proteins in the SDS-PAGE were transferred to a polyvinylidene difluoride membrane (Merck, New Jersey, USA). The membrane was subsequently incubated with 5% BSA for 2 h, followed by incubation with primary antibodies against E-cadherin (1:800, Abcam, Cambridge, UK), N-cadherin (1:800, Abcam, Cambridge, UK), PKA (1:800, Abcam, Cambridge, UK), vascular endothelial growth factor (VEGF; 1:800, Abcam, Cambridge, UK), EphA2 (1:800, Abcam, Cambridge, UK), MMP-2 (1:800, Abcam, Cambridge, UK), and GAPDH (1:800, Abcam, Cambridge, UK) overnight. After incubation in a solution of secondary antibody (1:2000, Abcam, Cambridge, UK) for 1.5 h, the membrane was exposed to ECL solution (Invitrogen, California, USA), and the relative expression level of target proteins was confirmed by visualization with Image J software [21 (link)].
6
Immunoprecipitation and Western Blotting
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Transfected HEK293T cells were homogenized using lysis buffer (TBS + 10% glycerol +1% Triton X-100) with protease inhibitors. Proteins were captured using anti-HA affinity gel (Biotool) at 4°C for 3 hours. Beads were washed several times using lysis buffer, and proteins were eluted using sodium dodecyl sulfate sample buffer.
Immunoprecipitation samples were separated using 12% polyacrylamide gels and transferred to the PVDF membranes (Millipore). Membranes were blocked using blocking solution (TBS + 0.05% Tween-20 + 5% skim milk) for 30 min. Proteins were detected using anti-DDDDK (Abcam) and anti-HA (Santa Cruz Biotechnology). Visualization was performed using HRP-conjugated antibodies (Invitrogen) with ECL solution (Invitrogen). Images were captured using ChemiDoc MP (Bio-Rad).
Immunoprecipitation samples were separated using 12% polyacrylamide gels and transferred to the PVDF membranes (Millipore). Membranes were blocked using blocking solution (TBS + 0.05% Tween-20 + 5% skim milk) for 30 min. Proteins were detected using anti-DDDDK (Abcam) and anti-HA (Santa Cruz Biotechnology). Visualization was performed using HRP-conjugated antibodies (Invitrogen) with ECL solution (Invitrogen). Images were captured using ChemiDoc MP (Bio-Rad).
7
Quantification of RNA-related Proteins
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The protocol of western blot was shown before [18 (link)]. Briefly, total protein was extracted by RIPA buffer, and centrifuged at 16,000 × g for 20 min, the supernatant was taken for protein quantification. Then, 30 μg protein was used for sample loading, and transferred onto PVDF membrane, followed by incubation with HK2 antibody (ab104836, Abcam), YTHDF1 (ab220162, Abcam), METTL3 (ab195352, Abcam) and GAPDH (ab2735, Abcam) overnight at 4°C. The second day, the membrane was incubated with HRP-conjugated IgG at 37°C for 1 h. After washing with TBST, the blot was developed using ready-to-use ECL solution (Invitrogen).
8
Western Blot Analysis of Apoptosis and Cell Cycle Markers
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After cell treatment, the medium was discarded. The protein lysis solution (Roche) was added, and the total protein was separated. Then, 50 μg of the total protein was taken and sampled on a 12% polyacrylamide gel and electrophoresed (100 V, 2 h). Afterward, the protein was transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skimmed milk at RT for 1 h, the membranes were rinsed with TBST 3 times (10 min each time) and incubated with the primary antibodies (1:1000) including anti-Bad (ab32445), anti-bcl2 (ab32124), anti-Bax (ab32503), anti-Caspase3 (ab32351), anti-p21 (ab109520), anti-Cyclin D (ab16663), anti-CDK4 (ab108357), anti-Cyclin E (ab33911), anti-CDK2 (ab32147), anti-GRIM-19 (ab110240), anti-E-cadherin (ab16505), anti-Vimentin (ab92547), anti-N-cadherin (ab18203), anti-p-STAT3 (ab76315), anti-STAT3 (ab68153), and anti-HIF-1α (ab179483) overnight at 4℃. After washing the membranes with TBST, we incubated them with horseradish peroxidase (HRP)-labeled goat-anti-rabbit IgG at RT (ab205718, 1:2500) for 1 h. The above antibodies were all from Abcam (Cambridge, UK). The membranes were rewashed 3 times with TBST (10 min each time). Finally, the ECL solution (Invitrogen) was utilized for protein band development and imaging, and Image J was employed to analyze the gray value of each protein.
9
Protein Extraction and Western Blot
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Total protein was extracted by RIPA buffer, and centrifuged at 16,000×g for 20min, the supernatant was taken for protein quanti cation. Then, 30µg protein was used for sample loading, and transferred onto PVDF membrane, followed by incubation with HK2 antibody (ab104836, Abcam), YTHDF1 (ab220162, Abcam), METTL3 (ab195352, Abcam) and GAPDH (ab2735, Abcam) overnight at 4 °C. The second day, the membrane was incubated with HRP-conjugated IgG at 37°C for 1h. After washing with TBST, the blot was developed using ready-to-use ECL solution (Invitrogen).
10
Exosome Protein Extraction and Western Blot
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RIPA lysis buffer (Lab-bio, China) added with the PMSF (Yuanye, China) were injected into the OC cells and exosomes. After ice bath for 30 min, the lysates were centrifuged at 12000×g and 4℃ for 8 min. The concentration and quality of the extracted protein was determined by spectrophotometry method. Then 15 μg of the protein was added to the loading well of 12% prefabricated gel and the electrophoresis was conducted. After that, the separated proteins in the gel were transferred to the nitrocellulose (NC) membrane (Millipore, USA). The NC membrane was soaked in the blocking buffer (1% BSA diluted by TBST) for 1 h. Then the NC membrane was immersed in different primary antibodies at 4℃ for overnight. After rinsing with TBST, the NC membrane was immersed in the secondary antibody solution for another 1 h. In the end, ECL solution (Invitrogen, USA) was dropped onto the membrane, and then development was performed with Bio-Rad gel imaging system (USA). Antibodies used in this research were all purchased from Abcam (UK), including anti-TSG101 (ab228013, 1:2000), anti-CD63 (ab321975, 1:1000), anti-GRP94 (ab3674, 1:3000), anti-Bax (ab263897, 1:2500), anti-Bcl2 (ab194583, 1:1500), anti-Cleaved Caspase-3 (ab231289, 1:800), anti-Cleaved Caspase-9 (ab2324, 1:800), anti-MMP2 (ab181286, 1:1000), anti-MMP9 (ab283575, 1:1500), goat anti-rabbit IgG (ab97051, 1:40000).
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