The trigeminal ganglia from mice were first homogenized by a tissue homogenizer, and then samples were lysed by RIPA (Beyotime, China) containing 1% PMSF (Solarbio, China). The cells were directly lysed by RIPA with 1%PMSF. The proteins were boiled at 100 °C for 10 minutes. The proteins were isolated by 10% SDS-PAGE gel (Shanghai Epizyme Biomedical Technology Co., Ltd., China) and transferred to polyvinylidene fluoride (PVDF) membrane (Solarbio, China). After blocking with 5% skim milk, PVDF membrane was incubated with primary antibody overnight at 4 °C. The primary antibodies used in this experiment mainly include Runx2 (Abcam, ab192256, 1:1 000), Sema3A (Abcam, ab23393, 1:1 000), NGF (Huabio, ET1606-29, 1:1 000), Rock2 (Huabio, ER1706-48, 1:500), Drp1 (Abcam, ab184247, 1:1 000), Mfn2 (Huabio, ER1802-23, 1:1 000), and GAPDH (Huabio, ET1601-4, 1:2 000). HRP-conjugated secondary antibody (1:2 500, Huabio) was used to combined primary antibody. Super ECL Plus kit (US Everbright, China) was used to visualize proteins, and images was obtained by a Chemi Doc Touch Imaging System (Bio-RAD, USA).
Super ecl plus kit
Manufactured by US Everbright
Sourced in China, United States
The Super ECL Plus kit is a chemiluminescent detection system used for the quantitative analysis of proteins in Western blotting applications. The kit contains reagents that produce a luminescent signal when combined with the target protein, allowing for the visualization and quantification of the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Solarbio (1 mentions)
Hrp conjugated secondary antibody, by Huabio (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Ab192256, by Abcam (1 mentions)
Ab184247, by Huabio (1 mentions)
Bicinchoninic acid kit, by Beyotime (1 mentions)
β actin, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Rabbit anti human β actin, by Proteintech (1 mentions)
Hrp conjugated affinipure goat anti rabbit secondary antibody, by Proteintech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Chemiscope 3000 mini, by Clinx (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Jnk1 2 3, by ABclonal (1 mentions)
Cox 2, by Bioworld Technology (1 mentions)
P jnk, by Bioworld Technology (1 mentions)
4 protocols using super ecl plus kit
1
Molecular Profiling of Trigeminal Ganglia
2
Ovarian Protein Extraction and Western Blot
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RIPA buffer (Tris 50 mmol/l, NaCl 150 mmol/l, EDTA 0.5 mmol/l, NP40 1%, Triton X-100 0.5%, glycerin 10%) supplemented with protease inhibitor cocktail (MCE, Shanghai, China) and phosphatase inhibitors was used to extract proteins from cells and fresh ovarian tissues. After using Bicinchoninic Acid Kit (Beyotime, Shanghai, China) for protein quantification, the lysate was metallized with added loading buffer at 100°C for 10 min to denature. The protein was transferred to the PVDF membrane after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. After blocked with 5% defatted milk powder dissolved in TBST (pH 7.4, NaCl 8 g/l, KCl 0.2 g/l, Tris 3 g/l) at room temperature for 1 h, the membrane was incubated with the primary antibody at 4°C overnight, including STMN2 (1:1000, ab185956, Abcam, U.S.A.) and β-actin (1:1000, A5441, Sigma, U.S.A.). The membrane was incubated with horseradish peroxidase-coupled secondary antibody at room temperature for 1 h after washed with TBST for four times. Target protein bands were developed by Super ECL Plus kit (US Everbright INC, U.S.A.) and quantitatively analyzed by Image J.
3
Western Blot Analysis of KDM2A Protein
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The cells were lysed in cold RIPA Lysis Buffer (Beyotime, Shanghai, China), and the protein concentration was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). After boiling in loading buffer at 95 °C for 5 min, 40 μg per lane of proteins were separated by 8% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked using 5% (W/V) non-fat milk in Tris-buffered saline containing 0.1% (V/V) Tween-20 at room temperature for 1 h and then incubated overnight at 4 °C with the primary antibodies: rabbit anti-human KDM2A (1:1,000; Abcam, Cambridge, MA, USA) and rabbit anti-human β-actin (1:3,000; Proteintech, Rosemont, IL, USA). After several washings, the membranes were then incubated with HRP-conjugated affinipure goat anti-rabbit secondary antibody (1:5,000; Proteintech, Rosemont, IL, USA) for 45 min at 37 °C. The protein bands were visualized using a super ECL plus kit (US Everbright, Suzhou, China). The levels of protein expression were evaluated by ImageJ software (version 1.41; National Institutes of Health, Bethesda, MD, USA).
4
Regulation of Inflammatory Pathways in RAW264.7 Cells
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RAW264.7 cells (1 × 106/well) were cultured in a 6-cell plate overnight and then pretreated with E9OAEE (6.25, 12.5, 25, 50 µg/mL) for 2 h and stimulated with LPS (1 µg/mL) for 24 h or indicated time, respectively. Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) on ice for 30 min, and the supernatant was collected. Proteins were quantified using Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China), and equal amounts of protein (40 µg) were separated via 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transferred onto PVDF membranes (Millipore, CA, USA). The membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies specific for iNOS (ABclonal, Wuhan, China), GAPDH (Bioworld Technology, Inc, MN, USA), COX2 (Bioworld Technology, Inc, MN, USA), JNK1/2/3 (ABclonal, Wuhan, China), p-JNK (Bioworld Technology, Inc, MN, USA), P38 (ABclonal, Wuhan, China), p-P38 (Bioworld Technology, Inc, MN, USA), ERK1/2 (ABclonal, Wuhan, China), and p-ERK1/2 (ABclonal, Wuhan, China). The membrane was then incubated for an additional 60 min with a goat anti-rabbit lgG/HRP (Bioss, Beiing, China). Then, the membrane was developed using Super ECL Plus kit (US Everbright Inc, Suzhou, China) for imaging with ChemiScope 3000 mini (Clinx, Shanghai, China).
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