Dulbecco's modified Eagle's medium (DMEM) and penicillin/streptomycin were purchased from Hyclone (Logan, USA). Foetal bovine serum (FBS) was purchased from Gibico (South America). HIF-1α antibody was purchased from Abcam (Cambridge, United Kingdom). VEGF antibody was purchased from RD (Minneapolis, USA). Antibodies against AKT, p-AKT, mTOR, p-mTOR, ERK, p-ERK, MMP2, and MMP9 were purchased from CST (Boston, USA). Horseradish peroxidase- (HRP-) conjugated secondary antibodies, fluorescent-conjugated secondary antibodies, New Super ECL Assay, and β-actin antibody were purchased from KeyGEN BioTECH (Nanjing, China). Transwell chambers and matrigel were purchased from Corning Life Sciences (8 μm pores, Tewksbury, MA, USA) and BD Biosciences (10.5mg/ml, San Jose, CA, USA), respectively. The QRT-PCR Kits were purchased from Takara (Shiga, Japan). The Cell Counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). The enzyme-linked immune sorbent assay (ELISA) kits for human VEGF were purchased from Multi Sciences (Shanghai, China). BCA Protein Assay Kits were purchased from Beyotime Biotechnology.
New super ecl assay
Manufactured by Keygen Biotech
Sourced in China
The New Super ECL Assay is a chemiluminescent detection system designed for the quantification of protein levels in Western blot analyses. It utilizes a proprietary enhanced chemiluminescent (ECL) substrate to generate a strong, stable luminescent signal that can be detected using standard imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Keygen Biotech (2 mentions)
Hif 1α antibody, by Abcam (1 mentions)
Qrt pcr kit, by Takara Bio (1 mentions)
Vegf antibody, by R&D Systems (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibodies, by Keygen Biotech (1 mentions)
Fluorescent conjugated secondary antibodies, by Keygen Biotech (1 mentions)
β actin antibody, by Keygen Biotech (1 mentions)
Matrigel, by Corning (1 mentions)
Transwell chamber, by Corning (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti histone h3 antibody, by Abcam (1 mentions)
Anti hmgb1 antibody, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protein extraction kit, by Keygen Biotech (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Anti bax, by Immunoway (1 mentions)
Anti yap taz, by Cell Signaling Technology (1 mentions)
Cell lysis buffer for western and ip, by Beyotime (1 mentions)
Anti yap1, by Abcam (1 mentions)
Anti pcna, by Immunoway (1 mentions)
4 protocols using new super ecl assay
1
Hypoxia Regulates Cell Signaling and Migration
2
Protein Expression Analysis in MCAO
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Cytoplasmic and nuclear protein extracted from the ipsilateral cortex tissues after 24 h of MCAO were lysed with a protein extraction kit (KeyGEN BioTECH, Nanjing, China) on ice and centrifuged at 16 000 × g at 4 °C for 15 min, and the quantity of protein was assessed by the BCA protein assay kit (KeyGEN BioTECH, Nanjing, China). Protein samples (30 μg each lane) were separated by 12% SDS-PAGE and then transferred onto PVDF membranes (Millipore, Shenzhen, China). The membranes were blocked with 5% fat-free milk powder in TBST buffer for 1 h and then incubated with anti-HMGB1 antibody (1:1000, Abcam, Cambridge, MA, USA), anti-β-actin antibody (1:1000, Abcam, Cambridge, MA, USA) and antihistone H3 antibody (1:1000, Abcam, Cambridge, MA, USA) at 4 °C overnight. Next, the membranes were incubated with the HRP-linked secondary antibodies for 1 h at room temperature. Protein signals were detected with Image Quant LAS 4000 mini system using a New Super ECL Assay (KeyGEN BioTECH, Nanjing, China).
3
Huaier Modulates Cellular Signaling
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Cells were treated with a series concentrations of Huaier (0, 5, 10 and 15mg/ml) for 24h, and then lysed by cell lysis buffer for Western and IP (Beyotime, P0013). Protein concentration was detected with BCA Protein Assay Kit (KeyGen BioTECH, KGP902). After that, 20µg proteins were separated by 10% or 12% SDS-PAGE and transferred onto Pure Nitrocellulose Blotting Membrane (Pall Corporation, P/N 66485). Then, the blots were blocked for nonspecific binding with 5% non-fat milk in PBST (PBS, Tween-20, pH7.4) at room temperature for 1h. Next, the blots were incubated overnight at 4℃ with 5% non-fat milk containing primary antibodies which are listed as follows: anti-PCNA (ImmunoWay, YM3031), anti-Ki-67 (ImmunoWay, YT2467), anti-β-actin (ImmunoWay, YM3028), anti-Bcl-2 (ImmunoWay, YM3041), anti-Bax (ImmunoWay, YT0455), anti-E-cadherin (ImmunoWay, YT1454), anti-N-cadherin (ImmunoWay, YT2988), anti-YAP1 (abcam, ab52771), anti-p-YAP1 (ab76252), anti-CyclinD1 (abcam, ab134175), anti-Cleaved Caspase Substrate Motif (Cell Signaling, #8698) and anti-YAP/TAZ (Cell Signaling, #8418). After that, incubating blots with secondary antibodies conjugated with HRP (Cell Signaling, #7074 or #7076) for 1h at room temperature. Finally, after washing three times with PBST, the blots were visualized by New Super ECL Assay (KeyGen BioTECH, KGP1128).
4
DNA Oligonucleotide Synthesis and Cell Line Experiments
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All DNA oligonucleotides (Supporting Information, Table S1 ) were synthesized and purified by Sangon (Shanghai, China). The DNA oligonucleotides were dissolved in 1 × Tank buffer (20 mM Tris,125 mM NaCl, 20 mM KCl, pH 7.5) and diluted in appropriate buffer prior to use or stored at −20 °C. DNA Marker, GoldView, SYBR Premix Ex TaqTM II and PrimescriptTM RT reagent Kit with gDNA Eraser were purchased from TaKaRa (Dalian, China). Dulbecco’s Modified Eagle Medium (DMEM) and Fetal Bovine Serum (FBS) were obtained from Gibco (Shanghai, China). CCK-8 assay was obtained from Dojindo (Shanghai, China). Phospho-HER2/ErbB2 Antibody Sampler Kit and β-Actin polyclonal antibody were obtained from Cell Signaling Technology (Shanghai, China). HRP anti-mouse and rabbit IgG were from Beyotime (Jiangsu, China).New Super ECL Assay was from KeyGen (Nanjing, China). RIPA (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton x-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, and leupeptin), phenylmethanesulfonyl fluoride (PMSF) and Bradford protein dye reagent were used in western blot. SK-BR-3and MDA-MB-231 (the human breast cancer cell lines) cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). All other reagents were of analytical grade, and Millipore-Q water (≥18 МΩ) was used in all experiments.
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