Lysate Preparation Protocol for Western Blotting

PhosphoSolutions’ step-by-step protocol shows how to properly lyse organs and tissue culture cells when focusing on preserving protein phosphorylation, including detailed differences in lysing adherent and suspension tissue culture cells. Within each section there is a detailed list of steps, buffers, and specific materials to help researchers through this process. Additionally, helpful technical tips are offered throughout the protocol to provide insight about various factors that should be considered when lysing organs and tissue culture cells. Our goal is to provide essential techniques or tools to help researchers obtain publishable and reproducible results.

Sample Preparation

Before you Begin:

  • Determine Protein Mass:
    Organs: Weigh fresh or thawed organs. If frozen, brush off frozen debris from organ. If fresh, wash organ with 1X PBS and dry with Kimwipe. Estimate protein mass as 10% of total mass.
    TC Cells: Make estimate based on confluency (adherent) and cell count (suspension).
  • Heat Block: Set to 95oC.
  • Sonicator: Select probe and optimize strength based on lysis buffer volume and tube size to avoid foaming.

Materials Required:

  • Lysis Buffer: 1%(w/v) SDS, 10 mM TRIS, 1 mM EDTA, pH 8.0
    *room temperature for organs
    *95˚C for adherent and suspension TC cells
  • 1X PBS: 137 mM NaCl, 28 mM Na2HPO4, 5.4 mM KCl, 2.9 mM KH2PO4, pH 7.6
  • Cell Scraper: For adherent tissue culture cells
  • Dissection tools, scalpel, spatula, scissors
  • Plastic transfer pipette
  • Conical tube or microtube with screwcap
  • Ice/water

Prepare Samples

A. Organs – Whole cell lysate

  1. Place organ in conical tube or microfuge tube with screw cap. For large organs cut into 1/4” sections. For organs that are thick and dense cut even smaller sections.
  2. Add lysis buffer prepared at room temperature. Choose a volume based upon desired concentration, which is based on the estimated mass. Keep sample at room temperature.
    Tech Tips:
    • 1% SDS lyses everything, preserving proteins from protease and phosphatase enzymes. Cocktail inhibitors are not necessary.
    • A concentration below 10 mg/mL is recommended for optimal lysing. Calculate concentration with formula below:

B. Adherent Tissue Culture Cells

  1. Wash cells with 2-10 mLs of room temperature 1X PBS once. Remove wash with transfer pipet.
  2. Add 95oC lysis buffer, enough to cover the entire plate.
    Tech Tips:
    • Recommended volumes: 2 mLs for 150 mm plate, 1 mL for 100 mm plate, 500 μl for 60 mm plate.
  3. Rock and rotate the plate to thoroughly coat cells.
    Tech Tips:
    • Adherent cells will immediately lyse and a glob of cells in lysis buffer will be present.
  4. Transfer to a conical tube or microfuge tube with screwcap.
    Tech Tips:
    • Tilt plate and gently guide lysed cells with cell scraper to collect at the bottom of the plate. Cut off the tip of a plastic transfer pipette to help collect and transfer the large glob of cells into conical tube.

C. Suspension TC Cells

    1. Pipette media and cells into conical tube.
    2. Pellet cells by centrifugation (1 minute at 1800 x g) and remove media.
    3. Resuspend cells with 1-3 mLs of room temperature 1X PBS to wash.
    4. Repeat step #2.
    5. Add 95oC lysis buffer.

Lyse Samples

  1. Sonicate sample in 5-20 second intervals until buffer is clear and can be easily pipetted without clogging.
    Tech Tips:
    For optimal lysing:
    • To prevent overheating, place conical or microfuge tube in an ice/water bath while sonicating. Do not keep sample in ice bath for an extended period of time.
    • To avoid puncturing/melting the plastic tube, minimize prolonged contact with the probe to the conical tube walls.
    • To avoid foaming, keep probe tip in lysis buffer until sonication is complete.
    • If the lysate sample clogs while pipetting, sonicate again until clog is no longer present.
  2. Heat sample at 95oC for 10 minutes. Cool to room temperature.
    Tech Tips:
    If lysate appears:
    • Cloudy: Add 5% by volume increments of lysis buffer to sample, then gently rock until sample is clear in solution. A protein concentration below 10 mg/ml is preferred. A concentration above this will appear cloudy and insufficiently lysed.
    • “Goopy”: Repeat sonication until sample is easily pipetted.
  3. Centrifuge lysate at 1800 x g for 5 minutes to pellet cell debris.
  4. Repeat steps 1-3 if debris is present. Place lysate in fresh conical or microfuge tube. Save/discard old tube and debris.
    Tech Tips:
    • Organ and cell membranes will appear as the white, stringy debris in lysate. It is essential to completely lyse the debris when studying transmembrane proteins.

Determine Protein Concentration

Determine protein concentration using BCA, Bradford, or Lowry assays.

Other Helpful Links:

Interested in buying our lysis buffer? You can!

Following all these steps and still ending up with goop? Check out our blog on The Truth about Goopy Lysate!

Read even more about SDS as a lysis buffer in our blog: 1% SDS is the Lysis Buffer of Choice for Most Western Blots

1% SDS Lysis buffer

Help ensure your lysing success by purchasing our pre-made lysis buffer. With 1% SDS, it will completely solubilize membrane and other hard to solubilize proteins, and even synaptic junction proteins!

Buy Our Lysis Buffer!

Help ensure your lysing success by purchasing our pre-made lysis buffer. With 1% SDS, it will completely solubilize membrane and other hard to solubilize proteins, and even synaptic junction proteins.

protein dephosphorylation

Protein Dephosphorylation Methods

This step-by-step method is broken down into 2 major sections on how to dephosphorylate proteins the right way. Within each section there is a detailed list of steps, buffers, specific materials, and calculation examples.

Western Blot Protocol

Broken down into 5 sections: sample prep, SDS PAGE, protein transfer, immunolabeling, and imaging, our WB protocol explains and highlights various factors that should be considered in your experiment.