Example Protocol: Cell Lysis for Downstream Protein Research

Cell lysis, also known as cell disruption, is a common laboratory technique aimed at extracting target nucleic acids or proteins inside the cell. In cell lysis or cellular disruption is a method in which the cell membrane is broken down or destroyed. In this process, subcellular components like DNA, RNA, proteins or organelles are freed from within the disintegrated cell membrane and become freely accessible in the working solution, termed the cell lysate. Cell lysis is an important first step in many research applications, including molecular diagnosis of pathogens, immunoassays for point of care applications, for studying protein function and structure, and in assessing drug response.

Varying cell lysis techniques are used across many fields (like research and development, clinical diagnostics, or industrial applications), for many types of organisms (including plant, animal, and bacteria), and many types of starting materials (for example tissues, cell suspensions, or whole blood). However for each cell lysis technique, the underlying goal remains the same: disrupt the cell membrane without causing damage to targeted intracellular components. The following protocol is geared toward the cell lysis of adherent cells or cells in suspension, for downstream protein research.

Equipment

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1. Sonicator or vortexer
2. Micropipette
3. Conical tube (sized accordingly for cell sample)
4. Microcentrifuge tube (sized accordingly for cell sample)

Reagents

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1. Mammalian cells grown in adherent (100 mm dish) or suspension culture
2. Ice cold RIPA Lysis Buffer
   1. A common lysis buffer that uses nonionic detergents and surfactants, like NP-40, and sodium dodecyl sulfate (SDS), and can be bought ready to use, or created in-house.
3. Protease and Phosphatase Inhibitor Cocktail (100X)
   1. A mix of inhibitors that is designed to prevent protein degradation by endogenous proteases and phosphatases released during the lysis process.
4. Ice cold PBS

Protocol

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1. Prepare the cells
   1. Adherent cells
      1. Culture adherent cells to ~80% confluence on a polystyrene tissue culture plate.
      2. Aspirate or decant excess media and keep the plate on ice.
      3. Wash the cell monolayer gently with 10 ml ice cold PBS. Aspirate excess PBS.
      4. Add 0.1 mL of protease inhibitors to 10 mL RIPA buffer to create the lysis buffer.
      5. Add 200 - 500 µl lysis buffer to the plate and gently swirl.
         1. The amount of lysis buffer should be empirically determined to ensure efficient lysis given the optimal desired concentration of protein in the lysate.
      6. Using a cell scraper or spatula, transfer the lysate to an appropriately sized conical tube.
   2. Suspension cells
      1. Culture cells to 1-2 x 10e6 cells/ml.
      2. Pellet the cells in a conical tube at 300 g for 5 minutes at room temperature.
      3. Aspirate or decant the media and keep cells on ice.
      4. Wash the pellet with 5 - 10 ml ice cold PBS.
      5. Centrifuge cells at 300 g for 5 minutes. Decant and aspirate the PBS excess supernatant, while keeping the pellet intact.
      6. Add 0.1 mL of protease inhibitors to 10 mL RIPA buffer to create the lysis buffer.
      7. Add 10 - 100 µl of lysis buffer per 1 x 10e6 cells.
         1. The amount of lysis buffer should be empirically determined to ensure efficient lysis given the optimal desired concentration of protein in the lysate.
2. Incubate the lysate on ice for 15 minutes.
3. Sonicate or vortex the lysate three times for 2 seconds each. Rest the lysate at least one minute in between pulses. Repeat as necessary if the lysate is still viscous.
4. Incubate the lysate on ice for 15 minutes.
5. Centrifuge the lysate at 13,000 g for 5 minutes at 4°C.
6. Collect the supernatant into new microcentrifuge tubes. Avoid disrupting the pellet
7. Aliquot and store the lysate at -20ºC.

It is also important to determine the protein concentration of the cell lysate before continuing to downstream applications. ELISA, for example, typically requires a starting protein homogenate concentration of 1-2 mg/mL. Alternatively for Western blot, the ideal protein concentration ranges between 1-5 mg/mL. This is generally done by either testing the spectrophotometric absorbance at 280 nm or through quantitative methods like the Bradford, Lowry, or BCA assays.

Tools:

Protein Concentration Calculator Bradford Protein Assay Calculator

Products

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References

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A Review on Macroscale and Microscale Cell Lysis Methods
Cell Disruption