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Protein extraction from TRIzol

This is a protocol for extracting protein from the TRIzol flow-through of an RNA extraction procedure (.e.g the DirectZOL kit from Zymo). The protocol is modified from instructions in the TRIzol user guide.

When best to use this protocol

  • When extracting RNA from TRIzol lysate in which the protein content may also be of interest.
  • When preparing lysate in which enzymatic degradation may be of significant concern (e.g. for phospho-Westerns).

When to use a different protocol

  • When native protein/RNA structure must be preserved (TRIzol denatures proteins and RNA).
  • When primary concern is maximum protein yield (it is difficult to completely recover proteins with this method).
  • When faster alternatives to protein extraction, like RIPA lysis buffer, are compatible with the experiment.

Reagents needed

  • 100% ethanol
  • 100% isopropanol
  • 6 M guanidine hydrochloride
  • 2X Laemmli buffer containing BME
  • 10% SDS

Procedure

  1. Lyse cells in ≥300 μL TRIzol per million cells.
  2. Homogenize lysate with pipetting or vortexing. This is important for efficient protein extraction—any goopy pellet will sequester the organic phase.
  3. Add an equivalent volume of 100% ethanol (e.g. add 300 μL 100% ethanol to 300 μL TRIzol sample), and mix by vortexing.
  4. Centrifuge through a Zymo IICR column (included with the Direct-zol kit). The RNA is bound to the column, and can be processed according to the Direct-zol kit instructions from this point.
  5. Add 1.5 volumes of 100% isopropanol to the flowthrough (e.g. add 900 μL 100% isopropanol to 600 μL TRIzol/ethanol flowthrough). An obvious precipitate should form when isopropanol is added. Mix by inversion and incubate for 10 minutes at room temperature.
  6. Centrifuge for 10 minutes at 4 °C, 12,000 g.
  7. During centrifugation, prepare a wash buffer of 19:1 100% ethanol and 6 M guanidine hydrocholride (e.g. combine 19 mL 100% ethanol and 1 mL GnHCl).
  8. Discard the supernatant in an appropriate waste container (the supernatant is 60% isopropanol, 20% ethanol, and 20% TRIzol).
  9. Add 1 mL of wash solution, vortex to resuspend pellets, and incubate for 20 minutes at RT on a rotating mixer.
  10. Briefly centrifuge pellets, remove supernatant, and repeat wash step two more times.
  11. After the third wash, centrifuge pellets and remove supernatant, then add 1.5 mL 100% ethanol, vortex to resuspend, and incubate for 5 minutes at RT on a rotating mixer.
  12. Briefly centrifuge pellets, completely remove supernatant, and air dry for 2-5 minutes. Be careful not to over-dry pellets!
  13. Prepare 1X Laemmli containing 1% SDS by combining 5 parts 2X Laemmli buffer, 1 part 10% SDS, and 4 parts water (e.g. combine 100 μL 2X Laemmli buffer, 20 μL 10% SDS, and 80 μL water).
  14. Add 100 μL of 1X Laemmli buffer with 1% SDS to pellets and incubate at 50 °C for 10 minutes.
  15. Vortex to resuspend pellet as completely as possible—it probably will not completely dissolve. Pipetting up and down also helps to resuspend as much of the pellet as possible.
  16. Incubate resuspended proteins at 100 °C for 5 minutes.
  17. Centrifuge and freeze at -20 °C (short term storage) or -80 °C (long term storage), until ready to run on a gel.