Adherent Cell Lysis Protocol
- 6 steps
- 1 hours
- 2 reagents required
Adherent cells present unique challenges due to their attachment to a surface, necessitating careful handling to ensure complete cell collection and effective lysis. This protocol outlines the steps required for cell preparation, focusing on preserving cellular component integrity. Special attention is given to maintaining the biological activity of proteins, nucleic acids, and other cellular constituents during the lysis process.
This protocol is intended for researchers in cell culture and molecular biology. It is adaptable to various cell types and downstream applications, providing a reliable approach to obtaining high-quality lysates from adherent cell cultures.
Reagents Required
| Product | Preparation |
| Phosphate Buffered Saline (PBS) | Use 10X PBS, pH 7.2 (0.2 M Potassium Phosphate, 1.5 M NaCl). Dilute appropriate volume to 1X with molecular biology grade water. |
| RIPA Lysis Buffer | Use 1X RIPA Lysis Buffer or prepare 1X solution with 10X RIPA Lysis Buffer and molecular biology grade water. |
Procedure
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Remove growth medium from the cells by decanting or aspirating the medium.
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Wash the cells with sterile PBS to remove residual medium. Slowly add a volume of sterile PBS equal to the original medium volume, being careful not to dislodge the cells. Mix gently and remove the wash solution. Repeat the wash once to remove any other minor contaminants. More washing steps can be done but two is usually sufficient to remove most of the contaminants.
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After removal of the final wash solution from the cells, add an appropriate volume of RIPA Lysis Buffer (1 mL for 0.5 to 5 x 107 cells). Incubate on ice or in a refrigerator (2°- 8°C) for 5 minutes.
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Rapidly scrape the plate with a cell scraper to remove and lyse residual cells. Transfer the cell lysate to a tube on ice. The lysate can either be used immediately or quick-frozen in liquid nitrogen and stored at –70°C for future use. It is best to freeze the lysate before clarification since the freeze-thaw cycle may cause some proteins to denature and aggregate.
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Clarify the lysate by centrifugation at 8,000 x g for 10 minutes at 4°C to pellet the cell debris.
Note: If a mucoid aggregate of denatured nucleic acids is present, carefully remove it with a micropipette before centrifugation. -
Carefully transfer the supernatant containing the soluble proteins to a tube on ice for immunoprecipitation or other analysis.