Ae. albopictus C6/36 cells (5×106 cells) were seeded into T-25 cell culture flasks. The cells were then infected with CHIKV-122508 at multiplicity of infection (M.O.I.) 10 and incubated at 37°C in the presence of 5% CO2 for 1.5 hours. The excess virus was washed off with 1X Phosphate Buffer Saline (PBS) and L-15 medium supplemented with 2% heat-inactivated FBS was added. After 24, 48 and 96 hours post-infection (h.p.i.), C6/36 cells were washed with 1 mL wash buffer thrice, provided from ProteoExtract Complete Mammalian Proteome Extraction Kit (Millipore) A cell scraper was used to scrap the monolayer of cells into the 1 mL of wash buffer. The cell pellet was obtained by centrifugation at 150×g for 10 minutes at 4°C and stored at -80°C for further analysis.
Proteoextract complete mammalian proteome extraction kit
Manufactured by Merck Group
The ProteoExtract Complete Mammalian Proteome Extraction Kit is a laboratory product designed to isolate and extract total cellular proteins from mammalian cells and tissues. The kit provides a comprehensive set of reagents and protocols to effectively separate proteins based on their cellular compartmentalization and solubility characteristics.
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2 protocols using proteoextract complete mammalian proteome extraction kit
1
CHIKV Infection Kinetics in Ae. albopictus C6/36 Cells
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Ae. albopictus C6/36 cells (5×106 cells) were seeded into T-25 cell culture flasks. The cells were then infected with CHIKV-122508 at multiplicity of infection (M.O.I.) 10 and incubated at 37°C in the presence of 5% CO2 for 1.5 hours. The excess virus was washed off with 1X Phosphate Buffer Saline (PBS) and L-15 medium supplemented with 2% heat-inactivated FBS was added. After 24, 48 and 96 hours post-infection (h.p.i.), C6/36 cells were washed with 1 mL wash buffer thrice, provided from ProteoExtract Complete Mammalian Proteome Extraction Kit (Millipore) A cell scraper was used to scrap the monolayer of cells into the 1 mL of wash buffer. The cell pellet was obtained by centrifugation at 150×g for 10 minutes at 4°C and stored at -80°C for further analysis.
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Ae. albopictus C6/36 cells (5×106 cells) were seeded into T-25 cell culture flasks. The cells were then infected with CHIKV-122508 at multiplicity of infection (M.O.I.) 10 and incubated at 37°C in the presence of 5% CO2 for 1.5 hours. The excess virus was washed off with 1X Phosphate Buffer Saline (PBS) and L-15 medium supplemented with 2% heat-inactivated FBS was added. After 24, 48 and 96 hours post-infection (h.p.i.), C6/36 cells were washed with 1 mL wash buffer thrice, provided from ProteoExtract Complete Mammalian Proteome Extraction Kit (Millipore) A cell scraper was used to scrap the monolayer of cells into the 1 mL of wash buffer. The cell pellet was obtained by centrifugation at 150×g for 10 minutes at 4°C and stored at -80°C for further analysis.
2
Protein Extraction from C6/36 Cells
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Cellular protein extraction of C6/36 cells was carried out with the use of the ProteoExtract Complete Mammalian Proteome Extraction Kit (Millipore) according to the instructions provided by the manufacturer. The cell pellets of different time points of CHIKV-infection (24, 48 and 96 h.p.i) were resuspended in an ice cold resuspension buffer. The cell pellet was then lysed using room temperature extraction reagent provided. Reducing agent and enzyme (Benzonase Nuclease) were added into the suspension to disrupt disulfide bonds and allow degradation of RNA and DNA. The fully solubilized suspension was centrifuged for 30 minutes at 16,000×g at 4°C and stored at -20°C. RC DC Bradford assay (BioRad) was used to determine the protein sample concentrations with BSA (0.5mg/mL) as the standard solution.
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