Protein was extracted using RIPA lysis buffer (Strong, YEASEN) and electrophoresis was performed using a PAGE Gel Quick Preparation Kit (10%, YEASEN) following the manufacturer's instructions. WB was carried out with the following antibodies: (primary) RARA (Invitrogen, #MA1-810A, 1:1000), RXRA (Invitrogen, #433900, 1:1000), HOXA1 (Invitrogen, #PA5-68809, 1:1000), FLAG (Beytime, #AF-0036, 1:1000) and GAPDH (Santa Cruz, #SC-365062, 1:2000), and then HRP-linked secondary antibodies (Sungene Biotech). HRP activity was detected using Luminol HRP Substrate (Millipore). Digital images were taken using an automatic chemiluminescence imaging analysis system (Tanon) (6 (link),26 (link),32 (link),33 (link)).
Page gel quick preparation kit
Manufactured by Yeasen
Sourced in China
The PAGE Gel Quick Preparation Kit is a laboratory equipment designed to facilitate the preparation of polyacrylamide gel electrophoresis (PAGE) gels. The kit provides the necessary components and instructions to efficiently create PAGE gels for various analytical and separation applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein quantification kit, by Yeasen (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Hrp linked secondary antibodies, by Sungene Biotech (1 mentions)
Ripa lysis buffer strong, by Yeasen (1 mentions)
Luminol hrp substrate, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
N 3 dimethylaminopropyl n ethylcarbodiimide hydrochloride edc hcl, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Skim milk, by Beyotime (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Yeasen (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Dimethyl sulfoxide (dmso), by Biosharp (1 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (1 mentions)
Gapdh, by ZenBio (1 mentions)
Trizol reagent, by Biosharp (1 mentions)
5 azacytidine, by Selleck Chemicals (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Crystal violet, by Beyotime (1 mentions)
6 protocols using page gel quick preparation kit
1
Protein Extraction and Western Blot Analysis
2
Multifunctional Nanoparticle Synthesis and Evaluation
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GO was purchased from XFNANO material Tech. Co., Ltd. (Nanjing, China). Iron (III) chloride hexahydrate (FeCl3·6H2O), ethylene glycol (EG), and sodium acetate (NaAc) were provided by Guanghua Tech. Co., Ltd. (Guangzhou, China). Six-armed polyethyleneglycol with six amino end groups (6-armed PEG–NH2) was obtained from Ponsure Biotech. Inc. (Shanghai, China). DMEM, trypsin, fetal bovine serum (FBS), and N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were offered by Sigma–Aldrich LLC. (St. Louis, MO, USA). Verapamil, tamsulosin, and valsartan were obtained from Ange Pharmaceutical Co., Ltd. (Nanjing, China). Vinorelbine ditartrate, fluorescein Isothiocyanate (FITC), coomassie brilliant blue R250, PAGE gel quick preparation kit, hoechst 33,258 stain solution, and annexin V-FITC/PI apoptosis assays kit were offered by Yeasen Bio. Tech. Co., Ltd. (Shanghai, China). Byakangelicol, imperatorin, and isoimperatorin were obtained from Herbest Bio. Tech. Co., Ltd. (Baoji, China).
3
Protein Analysis by Western Blotting
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Proteins were extracted with Nuclear and Cytoplasmic Protein Extraction Kit (Yeasen, China), and their concentrations were checked with BCA Protein Quantification Kit (Yeasen, China). After being separated using SDS-PAGE with PAGE Gel Quick Preparation Kit (12.5% and 10%) (Yeasen, China) and transferred onto the PVDF membrane (GE, USA), the unreacted sites were blocked with 5% skim milk (Beyotime, China) for 2 h and the primary antibodies against SLC7A5 (1 : 200, goat, ab99419), β-actin (1 : 500, goat, ab8229; 1 : 1000, rabbit, ab8227), p-mTOR (phospho-S2448, 1 : 1000, rabbit, ab109268), mTOR (1 : 1000, rabbit, ab32028), p-S6K1 (phospho-S424, 1 : 500, rabbit, ab131436), S6K1 (1 : 5000, rabbit, ab32529), p-4EBP (1 : 500, rabbit, ab47365), and 4EBP (1 : 2000, rabbit, ab32024) (Abcam, USA) were added and the samples were incubated at 4°C for 12 h. After being rinsed three times with TBST (Yeasen, China), the secondary HRP-conjugated donkey anti-goat antibody (1 : 1000, ab6885) or goat anti-rabbit antibody (1 : 2000, ab6721) (Abcam, USA) was administrated to the membrane and maintained for 1 h at room temperature. Next, the bands were washed with TBS-T and visualized using the 4CN HRP kit (Leagene, China). The relative expression levels were obtained via ImageJ 1.53f (NIH, USA).
4
Liposomal Transfection and Western Blot Protocol
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Hieff Trans Liposomal Transfection Reagent and PAGE Gel Quick Preparation Kit (12.5%) were purchased from Yeasen (Shanghai, China). Penicillin-streptomycin solution (100 ×), RIPA lysis buffer, and crystal violet were sourced from Beyotime (Shanghai, China). Fetal bovine serum (FBS) and RPMI-1640 medium were obtained from Bio-Channel (Nanjing, China). TRIzol reagent and dimethyl sulfoxide were purchased from Biosharp (Hefei, China). 5-Azacytidine was acquired from Selleck (Houston, United States of America). Paraformaldehyde was obtained from Servicebio (Wuhan, China). Cell counting kit-8 (CCK-8) was sourced from topscience (Shanghai, China). Nitrocellulose filter (NC) membranes were purchased from PALL (New York, United States of America). TMEM100 and β-actin primers were procured from Tsingke (Beijing, China). TMEM100 monoclonal antibodies were purchased from Proteintech (Wuhan, China). Human monoclonal antibodies against extracellular regulated kinase 1/2 (ERK1/2), phosphorylated (p-) ERK1/2, the c-Jun N-terminal kinase (JNK), phosphorylated (p-)JNK, p38, phosphorylated (p-) p38, goat anti-rabbit horse radish peroxidase (HRP) IgG, goat anti-mouse HRP IgG, and GAPDH were purchased from Zen Bioscience (Chengdu, China).
5
Western Blot Analysis Workflow
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The cells were lysed using RIPA buffer (Boster Biological Technology Ltd., Wuhan, China). The protein concentration was determined based on the BCA protein assay kit (Boster). Proteins were separated by uusing PAGE Gel Quick Preparation Kit (Yeasen Biotechnology, Shanghai, China), and the separated proteins were electro-transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was probed with diluted primary antibodies followed by overnight incubation at 4 °C. The next day, the membrane was labeled with HRP-bound secondary antibody incubation (1:1000, Beyotime, Shanghai, China) and detected using an ECL system (Bio-Rad, California, USA). The resulting bands were scanned using the ChemiDocTM MP Imaging System (Bio-Rad, California, USA).
6
Placental Protein Profiling in Normal and Pre-Eclampsia Pregnancies
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Proteins from placentas in women with normal pregnancy or PE (matched for gestational age, fetal sex and primiparity) and cell lines were extracted using a total protein extraction kit (Beyotime, China). The protein concentration was determined using a BCA protein quantification kit (Yeasen, China). All proteins were standardized to a concentration of 3 μg/μL and denatured at 100 °C for 5 minutes in SDS–PAGE protein loading buffer (5X) (Beyotime, China). The proteins were separated with a PAGE gel quick preparation kit (Yeasen, China) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Subsequently, the membranes were blocked with 5% skim milk powder (Solarbio, China) for 1 h and incubated with primary antibodies against CXCL3 (1:500, Sigma, USA), ERK1/2 (1:1000, ABclonal, China), p-ERK1/2 (1:1000, ABclonal, China) or β-actin (1:5000, ABclonal, China) overnight at 4 °C. Next, the membranes were washed three times with TBST and then incubated with a secondary antibody (1:100000, ABclonal, China) for 1 h at room temperature. Finally, SuperKine™ West Femto Maximum Sensitivity Substrate (Abbkine, China) was used to detect the chemiluminescence intensity with a ChemiDoc™ MP Imaging System (Bio-Rad, USA). Image Lab 6.0 software (Bio-Rad, USA) was used for gray value calculation. All experiments were conducted in triplicate.
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