Protein harvesting from cells typically involves protein extraction, which can be achieved through a combination of cell lysis, protein solubilization, and purification techniques. Here's a common protocol:
Protein Extraction Protocol
This protocol outlines a basic method for extracting proteins from cells. Specific modifications might be needed based on cell type and downstream applications.
1. Cell Harvesting
- Scrape: Using a cold plastic cell scraper, carefully detach cells from their growth surface. Cold temperatures help to minimize protein degradation.
- Collect: Transfer the scraped cells into microcentrifuge tubes.
2. Cell Lysis
- Agitate: Subject the cell suspension to agitation (e.g., vortexing, shaking) in the microcentrifuge tubes for 30 minutes at 4°C. This helps to break open the cells and release their contents, including proteins. Alternative lysis methods can include sonication, freeze-thaw cycles, or enzymatic lysis, often with commercially available lysis buffers. The choice depends on the cell type and the sensitivity of the target proteins.
3. Clarification
- Centrifuge: Centrifuge the tubes at high speed (16,000 x g) for 20 minutes at 4°C. This step separates the cellular debris (membranes, nucleic acids, etc.) from the soluble protein fraction.
- Collect Supernatant: Carefully remove the supernatant (the liquid above the pellet) containing the extracted proteins and transfer it to a new tube. This supernatant represents your crude protein extract.
Important Considerations:
- Protease Inhibitors: Add protease inhibitors to the lysis buffer and throughout the extraction process to prevent protein degradation by cellular proteases.
- Detergents: Lysis buffers often contain detergents (e.g., Triton X-100, SDS) to help solubilize membrane proteins. The choice of detergent depends on the protein of interest and downstream applications.
- Buffer Choice: Select a buffer with a pH suitable for the protein(s) of interest to maintain stability and activity. Common buffers include Tris-HCl and phosphate buffers.
- Temperature: Keep samples cold (4°C) throughout the process to minimize protein degradation and denaturation.
Further Steps (Optional):
- Protein Quantification: Determine the protein concentration of the extract using methods like Bradford assay, Lowry assay, or BCA assay.
- Protein Purification: Depending on the application, further purification steps may be necessary, such as affinity chromatography, ion exchange chromatography, or size exclusion chromatography.
This procedure provides a basic method for harvesting protein from cells. Adjustments to the buffer composition, lysis method, and subsequent purification steps should be made based on the specific needs of the experiment.
