For Western blotting, the total protein of each sample was measured by the BCA protein assay kit (Thermo Scientific, Rockford, USA). The protein from each sample was separated by 4–12% NuPAGE (180-8018H, Tanon). After transfer of proteins to polyvinylidene fluoride membrane (Millipore, Billerica, USA), the membrane was blocked with 5% skim milk for 1.5 h at room temperature and incubated for 2 h at room temperature with the primary antibodies: anti-VEGF antibody (1:1000, 19003-1-AP, Proteintech), anti-PI3K antibody (1:1000, 4257, Cell Signaling Technology), anti-p-PI3K antibody (1:1000, 4228, Cell Signaling Technology), anti-AKT antibody (1:1000, 4691, Cell Signaling Technology), anti-p-AKT antibody (1:2000, 4060 T, Cell Signaling Technology), anti-ERK antibody (1:1000, 4695, Cell Signaling Technology), anti-p-ERK antibody (1:2000, 4370, Cell Signaling Technology), and anti-GAPDH antibody (1:10000, 60004-1-Ig, Proteintech). Thereafter, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 1 h and treated with enhanced chemiluminescent reagent kit (Thermo Scientific, Rockford, USA). The bands were scanned and digitalized. The density of each band was quantified using ImageJ and normalized to the values of GAPDH.
Enhanced chemiluminescent reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Enhanced Chemiluminescent Reagent Kit is a laboratory product designed for the detection and quantification of proteins in Western blot analysis. The kit contains the necessary reagents to generate a chemiluminescent signal that can be captured and analyzed using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Fabp4, by Abcam (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Runx2, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab222754, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Hrp conjugated goat anti mouse igg h l, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
4 protocols using enhanced chemiluminescent reagent kit
1
Western Blotting Protein Quantification
2
Western Blot Analysis of Stem Cell Differentiation
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After being cultured in adipogenic or osteogenic medium, BMSCs were harvested and lysed with RIPA (Solarbio). Protein concentrations were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were denatured and resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore), incubated with 5% skimmed milk (Biofath) for 1–1.5 h at 25 °C, and then incubated overnight at 4 °C with primary antibodies. Primary antibodies were diluted as follows: PPARγ (1:500; Santa Cruz Biotechnology), Fabp4 (1:1000; Abcam), Runx2 (1:1000; Cell Signaling Technology), and ALP (1:1000; Santa Cruz Biotechnology). β-Actin (1:5000; Cell Signaling Technology) or α-Tubulin (1:10,000; Sungene Biotech) was used as loading control. Consecutively, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000; Cell Signaling Technology) dissolved in blocking buffer for 2 h at 25 °C. Blots were detected using an enhanced chemiluminescent reagent kit (Thermo Fisher Scientific).
3
Quantification and Western Blot Analysis of Protein Expression
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The total protein was extracted from the HOEC cell line or OSCC cell lines using radioimmunoprecipitation assay (RIPA) lysis buffer and was quantified with the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA). After that, 30 μg extracted protein was loaded into an 10% SDS-PAGE gel and separated by electrophoresis. Next, the protein bands were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA) and washed three times with PBST (0.1% Tween 20–phosphate-buffered saline [PBS] [vol/vol]). The membrane was then blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature, followed by primary antibody incubation overnight at 4°C. After washing the membrane three times with PBST, it was incubated in diluted secondary antibody at room temperature for 1.5 h. Finally, the blots were visualized with the Enhanced Chemiluminescent reagent kit (Thermo Fisher Scientific. USA). The primary antibodies used were rabbit monoclonal anti-SERPINE1 (1:1,000, catalog no. ab222754; Abcam, USA) and mouse monoclonal anti-GAPDH (1:1,000, catalog no. ab8245; Abcam, USA). The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG(H+L) (1:1,000, catalog no. ab205718; Abcam, USA) and HRP-conjugated goat anti-mouse IgG(H+L) (1:1,000; catalog no. ab6789; Abcam, USA).
4
Brain Microvascular Protein Analysis
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Brain microvascular segments were isolated from brain tissues by density gradient centrifugation with 15% dextran and stored at −80°C until needed. TGN lysis buffer (HEPES 50.0 mmol/L, NP-40 0.5%, Tween-20 1.0%, PMSF 1.0 mmol/L, NaF 1.0 mmol/L, NaVO3 1.0 mmol/L) was used for extraction of proteins from cultured cells and microvascular tissues of the rat brain. After blocking with 5% milk, blotting membranes were incubated overnight at 4°C with the primary antibodies for ZO-1 (1:1000), claudin-5 (1:1000), occludin (1:500), Akt (1:2000, 4685, Cell Signaling, Danvers, MA, USA), p-Akt (Ser473) (1:2000, 9271S, Cell Signaling), GSK-3β (1:1000, 9315, Cell Signaling), p-GSK-3β (Ser9) (1:1000, 9336S, Cell Signaling), β-catenin (1:1000, 8480T, Cell Signaling), and β-actin (1:1000, 4970S, Cell Signaling). The membranes were then washed, incubated with secondary antibodies, and processed with an enhanced chemiluminescent reagent kit (Thermo Fisher Scientific). The relative levels of the target proteins were analyzed by a densitometric method. The relative levels of claudin-5, ZO-1, occludin, and β-catenin were normalized to that of β-actin. The p-Akt and p-GSK-3β levels were normalized to those of total Akt and GSK-3β, respectively.
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