Cell lysates of A549 attached cells and spheres were prepared according to manufacturer's details of the protein extract kit (KEYGEN Biotech, Beijing, China). Protein concentrations were determined using the bicinchoninic acid method. Equal quantities of protein (50 μg/lane of total extract) were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then electrophoretically transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked in 5% skim milk powder dissolved in Tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature, following which they were incubated with the following primary antibodies at 4°C overnight: ABCG2, CD133 and β-actin. The membranes were then washed three times in TBST buffer, and were subsequently incubated with secondary antibody for 1.5 h at room temperature. The bands were visualized using a molecular imaging system. Densitometric analysis was performed using Image-ProPlus software (Media Cybernetics, Inc., Rockville MD, USA), and β-actin was used as a control.
Protein extract kit
Manufactured by Keygen Biotech
Sourced in China
The Protein Extract Kit is a laboratory tool designed to isolate and purify proteins from various biological samples. It provides a standardized and efficient method to extract proteins for further analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Dextran sulfate sodium salt, by Merck Group (1 mentions)
Ez ecl, by Sartorius (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
L serine, by Merck Group (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Cell death detection kit, by Roche (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Phospho pten ser380 thr382 383, by Cell Signaling Technology (1 mentions)
3 protocols using protein extract kit
1
Western Blot Analysis of Cancer Stem Cell Markers
2
Dextran Sulfate Sodium-Induced Colitis Model
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Dextran sulfate sodium salt (DSS) and L -serine were purchased from Sigma-Aldrich, (Shanghai, China). The animal diet was purchase from Research Diets, Inc. (New Brunswick, NJ, United States). Cell death detection kit was purchased from Roche (Shanghai, China). ELISA quantitative kits for the detection of IgA, IgG, IgM, IL-1β, IL-6, TNF-α, myeloperoxidase (MPO), and eosinophil peroxidase (EPO) concentrations were purchased from Cusabio Biotech (Wuhan, Hubei, China1 ). The QIAamp DNA stool MiniKit and Gel Extraction Kit were purchased from Qiagen (Hilden, Germany). The Protein Extract Kit was purchased from Keygen (Nanjing, China). Caspase 3, proliferating cell nuclear antigen (PCNA), mammalian target of rapamycin complex I (mTORC1), and general control non-derepressible 2 (GCN2) antibodies were purchased from Cell Signaling (Beverly, MA, United States). EZ-ECL was purchased from Biological Industries (Cromwell, CT, United States).
3
Myocardial Protein Extraction and Immunoblotting Analysis
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Protein extracts were prepared according to the manufacturer's protocol as described in the protein extract kit (Keygen Biotech Co., China). Peri-infarct region of the left ventricular myocardium was harvested after 2 h of reperfusion in order to extract total protein samples for immunoblotting analysis. Total protein was determined using the BCA Protein assay kit (Sigma-Aldrich, Germany), and size was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to PVDF membrane. Primary antibodies were then incubated with the membrane strips at 4°C overnight at the following dilutions: Akt, phospho-Akt (Ser473), PTEN, and phospho-PTEN (Ser380/Thr382/383) (Cell Signaling Technology, USA) 1 : 1000 and TOPK and phospho-TOPK (Thr9) (Abcam, UK) 1 : 1000. Then, protein bands were incubated with a secondary antibody and detected by the ECL chemiluminescence method. Densitometric analyses of western blot images were performed using ImageJ software (ImageJ 1.4).
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