The hippocampi of rats were rapidly collected and stored in liquid nitrogen immediately. The protein concentration was tested using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo scientific). Samples containing 40 μg of total protein were separated in 10%–15% sodium dodecyl sulfate (SDS)‐polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes (0.22 μm; Millipore Corp). The membranes were blocked in TBST containing 5% nonfat dry milk for 90 min and incubated with the following primary antibodies overnight at 4°C: anti‐PSD95 (goat monoclonal antibody; Abcam, 1:2,000), anti‐SYP (rabbit monoclonal antibody; Abcam, 1:2,000), anti‐GAP43 (rabbit monoclonal antibody; Abcam, 1:2,000), anti‐BDNF (rabbit monoclonal antibody; Abcam, 1:2,000), anti‐p‐CREB (phosphor S133, rabbit monoclonal antibody; Abcam, 1:5,000), anti‐CREB (rabbit monoclonal antibody; Abcam, 1:2,000), and anti‐β‐actin antibody (1:500, mouse monoclonal antibody; Boster, 1:500). On the next day, the membranes were incubated with the corresponding secondary antibody (anti‐goat/rabbit/mouse IgG; ZSGB‐BIO, 1:5,000) for 90 min at room temperature. After using clarity western electrochemiluminescence (ECL) substrate (Bio‐Rad), protein bands were visualized in the Bio‐Rad Imager. The intensities of the protein bands were analyzed by densitometry using Syngene imaging systems.
Clarity western electrochemiluminescence ecl substrate
Manufactured by Bio-Rad
Sourced in United States
Clarity western ECL substrate is a chemiluminescent reagent used for the detection of proteins in western blot analysis. The substrate emits light upon reaction with the enzyme-labeled detection antibody, allowing for sensitive and quantitative protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin antibody, by Boster Bio (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti syp, by Abcam (1 mentions)
Anti gap43, by Abcam (1 mentions)
Anti pcreb, by Abcam (1 mentions)
Imager, by Bio-Rad (1 mentions)
Anti creb, by Abcam (1 mentions)
Anti bdnf, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti psd95, by Abcam (1 mentions)
Imaging system, by Bio-Rad (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti vegfr2, by Abcam (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membranes 0.22 μm, by Merck Group (1 mentions)
Anti akt, by Abcam (1 mentions)
Sds page, by Merck Group (1 mentions)
6 protocols using clarity western electrochemiluminescence ecl substrate
1
Hippocampal Protein Expression Analysis
2
Hippocampal Protein Expression Analysis
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The hippocampus of each rat was rapidly dissected and stored in liquid nitrogen immediately after dissection. Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, USA). An equal amount (30 μg) of each sample was loaded on 6% and 10% SDS polyacrylamide gels. The proteins were separated by electrophoresis and then transferred to polyvinylidene difluoride membranes (0.22 μm, Millipore Corp, Billerica, MA, USA). The membranes were blocked with PBS containing 10% milk for 1 h and then incubated with the following specific primary antibodies overnight at 4°C: anti-VEGFR2 (rabbit polyclonal antibody, Abcam, 1:1000), anti-AKT (rabbit monoclonal antibody, Abcam, 1:2000), anti-phospho-akt (rabbit monoclonal antibody, Cell Signaling Technology, 1:2000), anti-ERK (rabbit monoclonal antibody, Cell Signaling Technology, 1:2000), and anti-phospho-erk (rabbit monoclonal antibody, Cell Signaling Technology, 1:2000). In parallel, the Western blots were probed with an anti-β-actin antibody (mouse monoclonal antibody, 1:500, Boster) for data normalization. The proteins were visualized using Clarity Western Electrochemiluminescence (ECL) Substrate (Bio-Rad, USA), and the gray values were analyzed using a Syngene imaging system.
3
Western Blot Protein Expression Analysis
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Tissues were homogenized in radioimmunoprecipitation (RIPA) lysis buffer. The supernatant was collected after centrifugation at 12,000 rpm for 30 min at 4°C. Total proteins (20–40 µg) were electrophoresed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride membranes (Millipore, MA, Boston, USA; Bio-Rad, CA, Berkeley, USA; New Life Science Products, NY, New York City, USA). Afterwards, the membranes were blocked with 10% nonfat dry milk, followed by incubation with primary and secondary antibodies. Membranes were detected by Clarity Western electrochemiluminescence (ECL) Substrate (Bio-Rad, USA) in conjunction with a chemiluminescence system (New Life Science Products, USA).
4
Comprehensive Protein Expression Analysis
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Initially, PC9 and A549 cells were treated with radioimmunoprecipitation assay (RIPA buffer, Beyotime, Shanghai, China) and quantitatively assessed by a bicinchoninic acid (BCA) protein detection kit (Beyotime, Shanghai, China). 10 µg of the extracted protein was loaded on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Sigma-Aldrich) and subjected to separation before being transferred onto polyvinylidene fluoride (PVDF) membranes. Further, 2.5% of skimmed milk was used to block the protein-blotted PVDF membranes. Afterward, the membranes were incubated with primary antibodies against DARS2 (1:1000), GAPDH (1:1000), AKT protein kinase B, (p-AKT 1:1000), p-AKT (1:1000), AKT (1:1000), phosphorylated-Phosphotylinosital 3 kinase (p-PI3K, 1:1000), and PI3K (1:1000) obtained from Abcam (Cambridge, USA). After incubation overnight, the horse-radish peroxidase (HRP)-conjugated secondary antibodies (1:1000, Abcam) were used to detect the protein blots. Finally, the protein signals were visualized through Clarity Western electrochemiluminescence (ECL) substrate (Bio-Rad, Hercules, CA, USA).
5
Synaptic Protein Quantification
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Total protein samples were extracted using a total protein extraction kit (BestBio) and the protein concentration was assessed using a Bio‐Rad protein assay kit (Bio‐Rad Laboratories). Samples containing 15 μg of protein were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (0.22 μm, Millipore). The membrane was blocked for 1 h at room temperature in PBS containing 5% bovine serum albumin and further incubated with specific primary detection antibodies (anti‐synaptophysin (SYP), rabbit monoclonal antibody (Abcam; 1:50,000); anti‐growth‐associated protein 43 (GAP‐43), rabbit monoclonal antibody (Abcam; 1:100,000); anti‐postsynaptic density protein 95 (PSD‐95), goat polyclonal antibody (Abcam; 1 μg/mL); and an anti‐β‐actin antibody (1:500, Chemicon)) overnight at 4°C. Subsequently, peroxidase–conjugated secondary antibodies were applied to the same membranes for another 1 h at room temperature. Protein bands were visualized using clarity western electrochemiluminescence (ECL) substrate (Bio‐Rad) and analyzed by densitometry using ACDSee Pro2.5 (ACDSee, Inc.) and Image J software (NIH).
6
Western Blot Analysis of Protein Extracts
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The L02 cells were lysed with ice-cold RIPA lysis buffer (65 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with a protease inhibitor (Roche, Basel, Switzerland) and a phosphatase inhibitor (Roche, Basel, Switzerland) and centrifuged at 12,000 rpm for 30 minutes at 4 °C. Total proteins (20–40 µg) were electrophoresed on SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore). Afterwards, the membranes were blocked with 10% nonfat dry milk, followed by incubation with primary and secondary antibodies. Membranes were detected by Clarity Western electrochemiluminescence (ECL) Substrate (Bio-Rad, USA) in conjunction with a chemiluminescence system (New Life Science Products, USA).
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