Total protein was extracted from tumor biopsies using Total Protein Extraction Reagent (Thermo Scientific, Waltham, MA) and complete protease inhibitor (Roche, Indianapolis, IN), then dissociated in a cold dounce homogenizer. Samples were disrupted by sonication, centrifuged to remove debris, and filtered through a 1.2μm Gelman 4190 syringe filter. Protein concentration was determined with a NanoDrop ND1000. Indicated cytokines and chemokines were measured in serum or biopsy samples using cytokine multiplex kits (EMD Millipore Corporation, Billerica, MA) and quantified against calibration curves from recombinant protein standards using a Bio-Plex array reader (Bio-Rad).
Total protein extraction reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Total Protein Extraction Reagent is a chemical solution designed for the extraction and isolation of total proteins from biological samples. It facilitates the efficient lysis of cells and the solubilization of proteins, enabling their subsequent analysis and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Cytokine multiplex kits, by Merck Group (2 mentions)
Bio plex array reader, by Bio-Rad (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Duosets, by R&D Systems (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Pierce 660 nm protein assay, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent kit, by Beyotime (1 mentions)
6 protocols using total protein extraction reagent
1
Tumor Protein Extraction and Cytokine Profiling
2
Protein Extraction from Perfused Lungs
3
Protein Extraction and Cytokine Quantification
4
Muscle Protein Extraction and Quantification
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Because swelling can occur in injured tissues, we measured the protein content of muscle tissues. Then 50 mg of finely minced muscle was placed in 500 µL of Total Protein Extraction Reagent (Thermo Fisher Scientific Inc., Rockford, Illinois) with 1:200 protease inhibitor cocktail (Thermo Fisher). Samples were homogenised, vortexed vigorously at 4º C, centrifuged at 10000 × g for ten minutes, and the supernatant was collected and stored at -80º C. Protein concentration was determined using a Bicinchoninic Acid protein assay (Thermo Fisher) and measured in a SpectraMax microplate reader (Molecular Devices, Sunnyvale, California). Relative protein concentration was determined by normalising the concentration from the BCA assay by the wet mass of muscle tissue.
5
Protein Extraction and Quantification
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Total protein extraction reagent (Thermo Fisher Scientific, Product No 78510) was used to extract protein from wound tissue according to the manufacturer’s instructions. Protein was quantified using a standard protein assay kit (Pierce™ 660 nm Protein Assay, Thermo Fisher Scientific). The optical density of the standard and unknown sample was measured at 660 nm using an ELISA plate reader (universal microplate reader) (Biotech Inc., Hawaii, USA). A standard curve was used to determine the protein concentration.
6
Quantitative Protein Expression Analysis
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Samples of uterine tissues were collected, and the total proteins were extracted by a total protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommended protocol. The concentration of the protein was measured with a bicinchoninic acid protein test kit (Thermo Fisher Scientific). Next, protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST) for 3 h at room temperature. Subsequently, the membranes were treated with specific primary antibodies (1:1,000 to 1:2,000 dilutions) at 4°C overnight, washed 3 times for 15 min each with TBST and secondary HRP-conjugated anti-mouse (1:10,000) or anti-rabbit (1:5,000) IgG antibodies and incubated at room temperature for 2 h. After 3 washes times with TBST, the blots were detected using an enhanced chemiluminescence reagent kit (Beyotime, China) and visualized with the Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA). All data are presented as the ratio of the target protein to the internal control (β-actin).
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