General info and notes before starting

Notes: Most of this protocol should be carried out on ice, unless otherwise indicated, to prevent Protein denaturation. Turn on the centrifuge before you get started so it can cool down.

Reagents

• Tri-Reagent (Invitrogen - AM9738)
• guanidine hydrochloride (Sigma - G3272-25G)
• Molecular grade ethanol
• Molecular grade isopropanol
• Molecular grade water
• 95% Ethanol (Histology grade)
• Lysis Buffer Solution (1.169g EDTA,1.636g NaCl,10g SDS, 2.4228 Tris, DI water to reach 200mL)

Protocol

1. See 1-4 of TriReagent RNA Isolation protocol. You will be using the pink bottom layer. Cut values in half if using 500 µL of TriReagent.
2. Thaw samples on ice.
3. Discard any remaining aqueous (colorless) phase.
4. Add 0.3 mL of 100% ethanol to the tube. Invert a few times and incubate at room temp for 2-3 min.
5. Centrifuge at 2000 x g for 5 min at 4°C to sediment DNA, then transfer pink supernatant to new tube. Discard the tube with the DNA pellet.
6. Precipitate protein by adding 1 ml of isopropanol, giving the tube a quick shake, and incubating for 10 min at room temp.
7. Centrifuge at 12000 rcf for 10 min at 4°C. Remove supernatant and discard as Trizol waste (toxic).
8. Wash pellet in 2mL of 0.3M guanidine hydrochloride 95% ethanol solution, let stand 20 min at room temp, then centrifuge at 7500 x g for 5 min at 4°C. Repeat this step 2 more times. (In this wash solution, the pellet should be stable for 1 yr @ -20°C.)
9. Vortex 3 sec in 1 mL 100% ethanol, let sit for 20 min at room temp.

10. Centrifuge at 7500 x g for 5 min at 4°C, remove supernatant and discard, let air dry 5 min. Dissolve pellet in Lysis buffer (amount depends on pellet size and desired concentration). To dissolve pellet, heat @ 65°C, occasionally vortexing, until dissolved.

    Congratulations, you’ve (hopefully) successfully isolated PRotein. You can now use if for applications like SDS-PAGE or Western Blotting.