Western Blot Protocol: Cell Lysis, Mammalian Cells

* For yeast and bacterial cell lysis consult Sambrook et al., or Harlow and Lane.

Adherent cells:

  1. Wash cells directly in the tissue culture flask or dish by adding cold PBS and rocking gently. Aspirate PBS and repeat. Keep tissue culture dish on ice throughout.
  2. Add appropriate volume ice cold lysis buffer (with fresh protease inhibitors), to the flask, approximately 1ml for a 100 mm tissue culture dish. (Alternatively, cells can be removed from the flask with Trypsin-EDTA and then prepared using the suspension cell instructions below).
  3. Incubate for 20 minutes on ice, and then scrape cells from the surface using a rubber spatula. 
  4. Transfer to a microfuge tube and clarify the lysate by spinning for 10 minutes at 12,000 rPM, at 4°C.
  5. Transfer supernatant to a fresh tube and store on ice or frozen at -20°C or -80°C.

 

Suspension cells:

  1. Pipet cells into a fresh conical tube and place on ice.
  2. Spin cells on low speed at 4°C, and aspirate off media.
  3. Add 10 ml ice cold PBS, and gently invert tube to wash cells.
  4. Spin cells on low speed, and aspirate off supernatant.
  5. Repeat wash and aspiration. resuspend cells in 5 ml ice cold PBS.
  6. Count cells, and centrifuge on low speed at 4°C to form a cell pellet. Aspirate off liquid.
  7. Gently resuspend the cell pellet in ice cold cell lysis buffer (with fresh protease inhibitors), use 1 ml buffer for 107 cells.
  8. Incubate cells for 30 minutes on ice. If needed, sonicate the lysates on ice for 15-30 seconds to disrupt genomic DNA and cellular components. 
  9. Transfer to a microfuge tube and clarify the lysate by spinning at 4°C, for 10 minutes at 12,000 RPM.
  10. Decant the supernatant to a fresh tube, and discard cell pellet. Store on ice for immediate use, or at -20°C or -80°C until needed.
  11. Measure protein concentration with BCA, Lowry, or Bradford assays, or by absorbance, prior to loading on a gel.

 

Tissue samples

  1. Tissue samples are rinsed in cold PBS and placed in a tube on ice immediately following dissection. 
  2. For small samples (100 mg) chop into pieces with clean dissecting instruments. Add tissue and 1-2 ml ice cold lysis buffer to dounce homogenizer, or sonicate in small tube. Homogenize or sonicate on ice.
  3. Clarify the lysate with a high speed spin in a microfuge at 4°C, for 10 minutes at 12,000 rPM.
  4. Transfer supernatant to a fresh tube and discard cell pellet. Store on ice for immediate use, or at -20°C or -80°C until needed.
  5. Measure protein concentration with BCA, Lowry, or Bradford assays, or by absorbance, prior to loading on a gel.

 

 


Chapter 5: Western Blot Buffers  Protocol: Gel Electrophoresis & Protein Transfer by Electroblotting