Aβ1-42 was purchased from GL Biochem (Shanghai) Ltd. (China) and was prepared as oligomers by incubating at a concentration of 1 mg/mL in sterile saline solution, followed by aggregation via incubation at 37°C for 4 days. In Situ Cell Death Detection Kit (11684817910) was purchased from Roche (Switzerland). Enzyme-Linked Immunosorbent Assay (ELISA) Detection Kits for brain derived neurotrophic factor (BDNF) (SEA011Mu) and nerve growth factor (NGF) (SEA105Mu) were purchased from USCN (China). RIPA lysis buffer and (P0013B), BCA Protein Concentration Kit (P0009) and ECL Plus reagent (P0018) were obtained from Beyotime Biotechnology (China). Antibody against GSK-3β (24198-1-AP) was purchased from Proteintech (China). Antibodies against phosphorylated GSK-3β (p-GSK-3β) (#5558), p-β-catenin (#9561), and Bax (#2772) were purchased from CST (USA). Antibodies against Tau (ab32057), p-Tau (ab109390), β-catenin (ab32572), cleaved caspase 3 (ab49822), Bcl-2 (ab196495), and doublecortin (ab18723) were purchased from Abcam (UK). β-actin (bsm-33036M) was purchase from Bioss (China). Secondary IgG-HRP goat anti-rabbit (A0208) and goat anti-murine (A0216) antibodies were purchased from Beyotime Biotechnology (China). Secondary Cy3-labled antibody goat anti-rabbit (A0277) was purchased from Beyotime Biotechnology (China).
Ecl plus reagent
Manufactured by Beyotime
Sourced in China
ECL Plus reagent is a luminescent detection system used for Western blotting applications. It is designed to provide a sensitive and quantitative means of detecting target proteins on nitrocellulose or PVDF membranes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (9 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Bca kit, by Beyotime (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti gapdh, by Abcam (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Pvdf membrane, by Beyotime (2 mentions)
Ab6721, by Abcam (2 mentions)
Bca assay kit, by Beyotime (2 mentions)
Ab49822, by Abcam (1 mentions)
Ab32057, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Ab109390, by Abcam (1 mentions)
β actin bsm 33036m, by Bioss Antibodies (1 mentions)
Ab32572, by Abcam (1 mentions)
33 protocols using ecl plus reagent
1
Amyloid-beta Oligomer Preparation and Analysis
2
Western Blot Analysis of DHCR7 in Cervical Cancer
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Cervical cancer cells were cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Inc., MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Inc., MA, USA). Cells were incubated at 37°C in a 5% CO2 humidified incubator. Protein lysis buffer (Beyotime, Shanghai, China) was used to extract the cell lysates and bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China) was used to determine the protein concentration. The protein samples were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (EMD Millipore, MA, USA). 5% bovine serum albumin (Beyotime, Shanghai, China) was used to block the membranes. Rabbit anti-DHCR7 antibody (1 : 2000, Invitrogen) and rabbit anti-β-actin were used to react with the protein samples at 4°C overnight. Then, goat anti-rabbit antibody (1 : 1000, Beyotime, Shanghai, China) was used to incubate the membranes at room temperature for 1 h. ECL plus reagent (Beyotime, Shanghai, China) was used to detect the brands.
3
Western Blot Analysis of FOXC1 Signaling
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Western blot analysis was performed as described previously (10 (link)). Total protein was extracted from SSC1 and SCC23 cells infected with lenti-shNC or lenti-shFOXC1 with RIPA lysis buffer (Beyotime Institute of Biotechnology). The concentration of total protein was measured with a BCA kit (Beyotime Institute of Biotechnology). Protein (10 µg/lane) was separated using 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) at 200 mA for 2 h. The blots were then blocked with 5% skimmed milk in TBS/T buffer for 1 h at room temperature, incubated overnight at 4°C with the primary anti-FOXC1 (cat. no. 8758; 1:500), anti-matrix metalloproteinase (MMP)-2 (cat. no. 40994; 1:800), anti-MMP-9 (cat. no. 13667; 1:500), anti-cyclin B1 (cat. no. 12231; 1:1,000), anti-cyclin D1 (cat. no. 2922; 1:1,000) and anti-GAPDH (cat. no. 51332, 1:5,000; all Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies, and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3,000) for 2 h at room temperature. Immunoreactive proteins were visualized using ECL Plus reagent (Beyotime Institute of Biotechnology). ImageJ software (version 1.8.0; National Institutes of Health, Bethesda, MD, USA) was used for densitometric analysis. Each assay was repeated 3 times.
4
Western Blotting of Xenograft and PDC Cells
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Western blotting was performed as our previous study [3 (link)]. The brief description is as follows. Freshly frozen xenograft tissue and PDC cells were lysed using 1% SDS solution (Beyotime), containing 1% phosphatase and proteinase inhibitor cocktail (Bimake). Approximately 200 mg of xenograft tissue was homogenized and lysed using 0.5 mL of lysis buffer. The protein concentration of individual samples was determined with BCA Protein Assay Kit (Beyotime). An equal quantity of protein was separated by 10% SDS-PAGE and transferred onto a transfer membrane (Millipore) and was blocked with 5% nonfat dry milk for 1 h at room temperature. After blocking with non-fat milk, the membranes were incubated with primary antibodies overnight at 4 °C: Rb (#ab24, Abcam), GAPDH (#2118, CST), phospho-Rb (S807/811, #8516, CST), CDK4 (#108,357, Abcam), CDK4 (#124,821, Abcam), and Cyclin D1(#55,506, CST). Horseradish peroxidase and secondary antibodies were used at room temperature for 1 h the following day. The chemiluminescence signal was developed with ECL-plus reagent (Beyotime) and detected by autoradiography.
5
Quantitative Analysis of TLR2, NF-κB, and Inflammasome Pathway Proteins
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We collected six sets of cell samples and lysed proteins with RIPA buffer (R0010, Solarbio, China) that contained Aprotinin and PMSF. Protein concentrations were determined by the BCA method, SDS-PAGE electrophoresis was performed using 7.5% and 12.5% separation gels (PG111, PG113, Epizyme Biotech, China), respectively, and the samples were transferred to PVDF membranes. Following blocking, we incubated the membranes with primary antibodies against TLR2 (Catalog No. 66645-1-Ig, Proteintech, China), P65 NF-κB (Catalog No. 10745-1-AP, Proteintech, China), p-P65 NF-κB (Cat.#: AF2006, Affinity Biosciences, China), NLRP3 (#M035175, abmart, China), IL-1β (Catalog No. 16806-1-AP, Proteintech, China), IL-18 (Catalog No. 60070-1-Ig, Proteintech, China), and β-actin (Catalog No. 20536-1-AP, Proteintech, China) at 4°C overnight. After washing in TBST, we incubated for 1 h using secondary HRP antibodies (Catalog No. SA00001-1, Catalog No. SA00001-2, Proteintech, China). The samples were exposed to a developer using ECL Plus reagent (P0018S, Beyotime Biotechnology, Shanghai, China). Grey-scale values were analyzed using Images-J to quantify protein expression levels based on band density.
6
Western Blot Analysis of FOXM1 Protein
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After the protein was extracted, the concentration was detected by a BCA kit (Beyotime). After denaturation, the protein was separated on 12% SDS-PAGE gels and then transferred to PVDF membranes (Beyotime). After blocking with 5% nonfat milk, membranes were incubated with the following primary antibodies: anti-FOXM1 (1:1000, Abcam, UK) and anti-GAPDH (1:1000, Abcam). After PBST washes, the membranes were incubated with an HRP-conjugated anti-mouse IgG antibody (1:2000, Beyotime, China) at room temperature for 1 h. Finally, the bands on the membranes were visualized with the ECL plus reagent (Beyotime) and photographed by a Tanon-5200 automatic chemiluminescence imaging analysis system (Tanon, Shanghai, China).
7
Protein Quantification and Western Blot
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Total proteins were extracted with RIPA buffer (Beyotime, Shanghai, China) following the manufacturer’s instructions. The protein lysate was electrophoresed and transferred to a PVDF membrane (Bio-Rad, CA, United States). The membrane was, respectively, incubated with the following primary antibodies from CST (Shanghai, China): p-PI3K (Tyr458/Tyr199; 1:1000; 4,228), PI3K (1:1000; 4,257), p-AKT (Ser473; 1:2000; 4,060), AKT (1:1000; 4,691), PTEN (1:1000; 9,559), survivin (1:1000; 2,808), FoxO1 (1:1000; 2,880), Bad (1:1000; 9,292), E-Cadherin (1:1000; 3,195) and N-Cadherin (1:1000; 13,116). The proteins were detected with ECL Plus Reagent (Beyotime, Shanghai, China) and quantified using ImageJ software (Yang et al., 2020 (link)).
8
Protein Expression Analysis in hESCs
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Proteins were extracted from cultured HESCs using RIPA lysis buffer, and the concentration of protein was detected by the BCA Protein Assay Kit (Beyotime). Samples were then separated by 10% SDS-PAGE gels (Beyotime). The primary antibodies were applied according to the provided recommendations: anti-ILK (1:5000, Abcam), anti-TGFβ1 (1:200, Boster), anti-SMAD2 (1:1000, Affinity), anti-VEGF (1:200, Boster), anti-COX-2 (1:700, Proteintech Group), anti-MMP-9 (1:800, Abcam) and anti-GAPDH (1:1000, Abcam). Finally, positive bands were detected using the chemiluminescent ECL Plus reagent (Beyotime) according to the manufacturer’s protocol. The densitometry of bands was quantified using Quantity One version 4.6.0 software (Bio-Rad). Expressions of proteins were normalized to GAPDH protein.
9
Protein Expression Analysis via Western Blot
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Total proteins were isolated by RIPA Lysis Buffer (Beyotime, Shanghai, China). Proteins’ concentrations were tested by bicinchoninic acid (BCA) kit (Beyotime). The protein sample was separated on SDS/PAGE gel, and transferred to polyvinylidene fluoride membranes, followed by the blockage with 5% nonfat milk for 1 h. Next, the membranes were probed with primary antibodies of ZDHHC5 (1:1000, Proteintech), Bcl-2 (1:1000, Abcam), Bax (1:1000, Abcam), pro-caspase-3 (1:1000, Abcam), cleaved-caspase-3 (1:1000, Abcam), pro-caspase-9 (1:1000, Abcam), cleaved-caspase-9 (1:1000, Abcam), COX-2 (1:1000, Abcam), and GAPDH (1:1000, Beyotime) overnight at 4°C. Then, membranes were incubated with secondary antibody (1:1000, Beyotime) for 2 h keeping in a dark place at room temperature. The protein levels were detected by enhanced chemiluminescence (ECL) Plus reagent (Beyotime).
10
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using RIPA buffer (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The protein lysate was subjected to electrophoresis and transferred onto a PVDF membrane (Bio-Rad, CA, United States). The following primary antibodies sourced from CST (Shanghai, China): p-MEK (Ser217/221; 1:1000; 9154), MEK (1:1000; 8727), p-ERK1/2 (Thr202/Tyr204; 1:2000; 4370), and ERK (1:1000; 4695), PD-1 (1:1000; 84651), PD-L1 (1:1000; 60475), and p-NF-κB (1:1000; bs-0982R) from Bioss (Beijing, China), NF-κB (1:1000; sc-8008) from Santa Cruz Biotechnology (Texas, United States) were used to incubate the membrane, respectively. The proteins were detected using ECL Plus Reagent (Beyotime) and quantified using Image Lab software.
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