Total cellular protein was extracted 48 h after transfection using protease‐containing RIPA lysis and phosphatase inhibitors. Proteins were then separated by electrophoresis using sodium dodecyl sulfate‐containing polyacrylamide gels and then transferred to polyvinylidene fluoride membranes. The membrane was blocked with 5% skimmed milk and incubated with primary antibodies (SMAD4 and glyceraldehyde 3‐phosphate dehydrogenase; Abcam) and horseradish peroxidase‐conjugated secondary antibody. The blots were visualized using an ECL Western blot kit (Millipore), following the manufacturers' protocol. The image was used for quantitative analysis of western blot bands obtained.
Ecl western blot kit
Manufactured by Merck Group
Sourced in United States
The ECL Western Blot Kit is a laboratory equipment product designed for the detection and analysis of specific proteins in biological samples using the Western blotting technique. It provides the necessary reagents and components required for the chemiluminescent detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bm0627, by Boster Bio (2 mentions)
Zb 2301, by ZSGB-BIO (2 mentions)
Ap124p, by Merck Group (2 mentions)
Bca protein assay kit, by Keygen Biotech (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Goat anti rabbit igg, by ZSGB-BIO (2 mentions)
Smad4, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Abcam (1 mentions)
Ab8448, by Abcam (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
Ab93876, by Abcam (1 mentions)
Ab93806, by Abcam (1 mentions)
Cw0096, by CWBIO (1 mentions)
Cw0103, by CWBIO (1 mentions)
Cw2333, by CWBIO (1 mentions)
Cw0014, by CWBIO (1 mentions)
Ab22552, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Bm0627, by Abcam (1 mentions)
9 protocols using ecl western blot kit
1
Western Blot Protein Quantification
2
Titanium Specimen Protein Expression Analysis
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The protein expressions of runt-related transcription factor 2 (Runx2) and osteopontin (OPN) on four different titanium specimens were analyzed by western blotting. After culturing cells (2 × 105 cells per well) on different specimens in 6-well plates for 7 and 14 days, the cells were rinsed with cold PBS and protein samples were harvested by lysis in RIPA buffer. The BCA protein assay kit (Key-GEN BioTECH, Nanjing, China) was used to determine total protein concentration. Protein extract samples (20 μg) were separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 hour at room temperature and then incubated with different primary antibodies against Runx2 (12556, CST, Beverly, MA, USA), OPN (ab8448, Abcam, USA), and β-actin (BM0627, Boster, Wuhan, China) overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibodies (ZB-2301, goat anti-rabbit IgG; ZSGB-BIO, Beijing, China; AP124P, goat anti-mouse IgG; Millipore, USA) for 2 hours. Finally, the membranes were visualized via ECL western blot kit (Millipore, USA). The protein expressions of Runx2 and OPN were evaluated relative to that of β-actin as the internal control. The experiments were performed in triplicate.
3
Osteogenic Potential on Titanium Surfaces
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Protein expressions of Runx2 and OCN of cells on titanium surfaces were examined by Western blotting. After culturing in 6-well plates on different samples pre-adsorbed with 0, 50, 100 and 200 μg ml−1 LDL for 3, 7 and 14 days, MC3T3-E1 cells (2 × 105 cells per well) were rinsed with cold PBS and protein samples were harvested by lysis in radio-immunoprecipitation (RIPA) buffer. The concentration of extracted total proteins was determined using the BCA protein assay kit (Key-GEN BioTECH, Nanjing, China). Protein samples (20 μg) were separated by electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies against Runx2 (12556; CST, Beverly, MA, USA), OCN (ab93876; Abcam, Cambridge, Ma, USA), and β-actin (BM0627, Boster, Wuhan, China) at 4 °C overnight. On the next day, the membranes were incubated with corresponding secondary antibodies (ZB-2301, Goat anti-Rabbit IgG, ZSGB-BIO, Beijing, China; AP124P, Goat anti-Mouse IgG, Millipore, USA) at room temperature for 2 h. After that, the membranes were visualized using the ECL Western Blot Kit (Millipore, USA). The protein expressions were determined relative to that of β-actin. The gray values of protein levels were quantified using the Photoshop software.
4
Circadian Rhythm Protein Quantification
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Protein concentrations were measured by the bicinchoninic acid assay (CW0014, CWBIO Company) after the total protein extracts were collected from pinealocytes using the radioimmunoprecipitation lysis buffer (CW2333, CWBIO Company). Equal amounts of protein from each sample were separated using SDS-PAGE and transferred onto PVDF membranes using electroblotting. The membranes were incubated in a solution containing 5% skim milk in phosphate buffer saline at room temperature for 1 h. The membranes were incubated with a CLOCK rabbit polyclonal antibody (1:1000, ab461, Abcam) and BMAL1 rabbit polyclonal antibody (1:1000, ab93806, Abcam) overnight at 4°C. After washing the membranes in Tris-buffered saline-Tween, they were incubated with goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin (Ig) G (1:8000, CW0103, CWBIO Company) for 1 h at room temperature. The blot bands were detected using the Ecl Western blot kit (1627003, Millipore) after washing. The intensity of the signals for CLOCK and BMAL1 were quantified using Image-Pro plus software and were normalised relative to the values obtained for β-ACTIN (1:4000, CW0096, CWBIO Company).
5
Osteogenic Differentiation Markers of MC3T3-E1 Cells on Titanium Alloy Surfaces
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The four materials (cp-Ti, Ti-SLA, Ti-NW and Ti-NW-Zn) samples were prepared and applied into 6-well plates. Subsequently, MC3T3-E1 cells (2 × 105 cells/well) were seeded onto each surface of alloy and cultured for 7 days. Protein samples were extracted from cells by RIPA buffer after cell washing with pre-cooled PBS. After the electrophoresis, protein samples were transferred to a polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at room temperature (5% nonfat dry milk, in Tris-buffered saline containing 0.1% Tween-20; TBST). They were subjected to incubation with primary antibodies against Runx2 (12556; CST, Beverly, MA, USA), OSX (ab22552; Abcam, Cambridge, MA, USA) or GAPDH (BM0627, Boster, China) overnight at 4 °C. After TBST wash for three times, membranes were incubated with secondary antibodies (ZB-2301; Goat anti-Rabbit IgG, ZSGB-BIO, China) for 2 h, followed by chemiluminescence exposure using ECL Western Blot Kit (Millipore, USA). The background was subtracted, and the signal of each target band was normalized to that of the GAPDH band. Protein level was quantified using the Image J software k1.45 (version k1.45; National Institutes of Health, Bethesda, MA, USA). The experiment was performed in triplicate.
6
Protein Extraction and Western Blot Analysis
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The total proteins extracted from the tissue were obtained using RIPA buffer (Beyotime, Shanghai, China), which contained PMSF (Beyotime) to inhibit protease degradation. Next, the samples were separated via electrophoresis on an SDS-PAGE gel (Solarbio) and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After that, the membranes were incubated overnight at 4 °C with one of the primary antibodies: γH2AX (BIOSS, Beijing, China) at a 1:500 dilution, CASPASE-3 (HUABIO, Hangzhou, China) at a 1:500 dilution, and β-actin (HUABIO) at a 1:5000 dilution. Next, a horseradish peroxidase-labeled secondary antibody (Thermo Fisher, Waltham, MA USA) was added and incubated at room temperature for 1 h. Finally, the protein bands were developed using an ECL Western Blot Kit (Millipore, Bedford, MA, USA), and the relative intensities of the bands were determined using Quantiscan software (Biosoft, Cambridge, United Kingdom).
7
Quantification of Inflammatory and Oxidative Markers
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The concentrations of serum IL-6 and TPO and the concentrations of reactive oxygen species (ROS), glutathione (GSH), and endostatin (ET) in brain tissues were measured by Sandwich ELISA according to the manufacturer’s instructions of the ELISA kit (LYBD Bio-Technique Co., Ltd., Beijing, China). For Western blotting, proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in blocking buffer [PBS, 0.5% Tween-20, and 5% non-fat dry milk powder or 3% bovine serum albumin (BSA)] and then incubated with primary antibody for 1 h at RT. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. The immunoreactive bands were visualized with enhanced chemiluminescence (ECL) Western blot kit (Millipore, Boston, MA, USA) and quantified using ImageJ version 1.50i.
8
Protein Extraction and Western Blot Analysis
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Total protein was extracted by lysing with RIPA buffer containing PMSF (1 mM) and phosphatase inhibitor (1 mM) mixture. Nuclear and cytoplasmic extracts were prepared using a kit (Cat. No. P0028, Beyotime Biotechnology) according to the manufacturer’s protocol. The expression of each protein was analyzed using western blotting according to a previously reported method [29 (link)]. Briefly, 30 μg of protein per well was detached by SDS-PAGE. The sample was transferred to PVDF membranes (Millipore). After blocking with 5% BSA for 1–2 h, the membranes were incubated with the diluted appropriate primary (1:1000) and HRP-conjugated IgG secondary (1:10000) antibodies. Signals were visualized using the ECL Western Blot Kit (Millipore).
9
Vascular Tissue Notch Signaling Analysis
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For Western blotting, mouse cervicothoracic vascular tissues (AOAR and CCA) were dissected from mice, weighted, and homogenized in RIPA buffer [50 mM Tris (pH7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS] (Boster Wuhan, China) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). The samples were centrifuged at 8,000 × g for 15 min at 4°C, and tissue debris was removed. Protein concentration was determined by BCA assay reagent (Pierce, Rockford, IL, USA). Proteins were separated by SDS-PAGE under reducing conditions and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in blocking buffer (PBS, 0.5% Tween 20, and 5% non-fat dry milk powder) and then incubated with primary antibody (anti-Notch3 NICD, anti-Hes5, anti-Hey2, and anti-GAPDH) for 1 h at RT (details of the dilution are described in the Table S1 ). After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. The immunoreactive bands were visualized with enhanced chemiluminescence (ECL) Western blot kit (Millipore, Boston, MA, USA) and quantified by ImageJ (version 1.50i).
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