A Dohlman Lab Protocol


 
 

TCA Whole Cell Extracts



REFERENCE: Adapted from Cox et. al. (1997) Mol. Biol. Cell, 8(9): p1805-1814.

Yeast proteins can be isolated efficiently and directly from intact cells by acid extraction using a 10% TCA solution.  Because of the very low pH of the TCA solution, the proteins are not readily degraded by proteases during the purification procedure.  

  1. Collect cell pellet for extraction by centrifugation at 4˚C.  Freeze cell pellet in liquid nitrogen and store at -70˚C.
  2. Typically 10 mL of early-log phase (OD600nm 0.6-1.0) culture should be used for the starting material, but smaller culture volumes can be used.  Where this is so, the final volume of resuspension buffer used to resuspend the protein pellet should be scaled down accordingly.
  3. To frozen pellet add 300 uL of TCA buffer, on ice. When thawed add half volume of glass beads and vortex using a multi-vortexer in 5 x 1 min bursts.  Chill tubes on ice for 3 mins between vortexing to keep cells chilled.
  4. The resuspension and wash volumes can be varied depending on your preference.  As proteins will be pelleted later on, larger volumes of TCA buffer and beads can be used to break open cells if desired.  If the cell pellet is quite large, using too small a volume at this step will reduce the overall yield of protein.
  5. Transfer the cell lysate to a new microfuge tube on ice.  Optional: Add 100 uL of fresh TCA buffer to the original microfuge tube that contains the glass beads.  Vortex briefly to wash the beads and pool this wash with the first cell lysate.  
  6. Centrifuge for 10 min, 16,000 x g at 4˚C to pellet the precipitated proteins and cell debris.
  7. Remove the supernatant by aspiration and resuspend the pellet in 150 uL of resuspension solution.
  8. Boil the samples for 5 mins, then allow to cool at room temp for a few minutes.
  9. Centrifuge the sample for 30 sec at 16,000 x g to pellet the cell debris.  Transfer 120 uL of the supernatant to a fresh microfuge tube. Measure protein concentration for 15 uL using the Bio-Rad DC Protein Assay kit for samples that contain detergent. Add 2x SDS-PAGE sample buffer to the remainder of the supernatant, boil for 5 min, and load SDS-PAGE gel. Samples can be stored at -20˚C but there may be some degradation of the sample.

TCA Buffer

10 mM Tris.HCl, pH 8.0
10% Trichloroacetic acid (TCA)
25 mM NH4OAc
1 mM Na2EDTA

This solution is best prepared by dilution of stock solutions.  For 100 mL add 1 mL 1 M Tris.HCl [pH 8.0], 10 mL 100% TCA solution, 1.67 mL of 1.5 M NH4OAc and 200 uL of 0.5M Na2EDTA.  Make up to 100 mL with milliQ water.  The pH of this solution will be in the range of 0.5-1.5 and does therefore not need to be sterilized.  It is also very corrosive.

Resuspension Solution

0.1 M Tris.HCl, pH 11.0
3% SDS

This solution can also be made up from stock solutions.  For 50 mL of solution, add 5 mL of 1M Tris.HCl [pH 11.0] and 7.5 mL of 20% SDS or alternatively 15 mL of 10% SDS.  Make up to 50 mL with milliQ water.  This solution can be filter sterilized if desired.

Antibody detection

p44/42 polyclonal antibodies from Cell Signaling Technology (#9101L) at 1:500 - 1:1000
p38 polyclonal antibodies from Cell Signaling Technology (#9211L) at 1:500 - 1:1000
(alternatively use p38 monoclonal antibodies from Cell Signaling Technology (#9216L))


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