Yes, Pamela Cayuleo there are alternative approaches for protein extraction from TRIzol-treated samples that do not require guanidine hydrochloride. While TRIzol is primarily optimized for RNA isolation, its protocol does allow sequential extraction of DNA and proteins. If guanidine hydrochloride is unavailable, you can still proceed with protein recovery using modified precipitation strategies. Here’s a reliable workaround:

Alternative Protocol for Protein Extraction from TRIzol Suspensions (Without Guanidine HCl)

1. Phase Separation:

• After homogenizing your cells in TRIzol, add chloroform (0.2 mL per 1 mL TRIzol), shake vigorously, and centrifuge at 12,000 × g for 15 minutes at 4°C.

• This separates the mixture into aqueous (RNA), interphase (DNA), and organic (protein) phases.

2. Protein Precipitation:

• Carefully remove the aqueous and interphase layers.

• To the remaining organic phase, add isopropanol (1.5 mL per 1 mL TRIzol used initially).

• Mix and incubate at room temperature for 10 minutes, then centrifuge at 12,000 × g for 10 minutes at 4°C.

3. Washing the Protein Pellet:

• Wash the pellet with 0.3 M guanidine acetate in 95% ethanol (if available), or alternatively, use 100% ethanol or acetone to remove residual phenol and salts.

• Repeat wash 2–3 times, centrifuging each time at 7,500 × g for 5 minutes.

4. Solubilization:

• Air-dry the pellet briefly (avoid over-drying).

• Resuspend in 1% SDS, 8 M urea, or RIPA buffer depending on downstream applications.

• Sonication or heating (e.g., 50–60°C for 10 minutes) may help solubilize stubborn pellets.

Tips for Optimization:

• Protein yield and integrity may vary depending on cell type and buffer choice. SDS is excellent for denaturing proteins for Western blotting, while urea is better for mass spectrometry.

• If downstream applications require native proteins, consider skipping TRIzol and using a dedicated protein extraction buffer instead.

While guanidine hydrochloride enhances protein solubilization, it’s not strictly necessary. With careful phase separation and ethanol-based precipitation, you can recover proteins efficiently from TRIzol-treated samples. Just tailor your solubilization buffer to your analytical needs.

TRIzol allows sequential extraction of RNA, DNA, and proteins, but the protein recovery step usually requires guanidine hydrochloride (or guanidine thiocyanate) to solubilize and wash the protein pellet. If guanidine hydrochloride is unavailable, you still have a few options:

• Use guanidine thiocyanate (if available), which is functionally similar for washing TRIzol protein pellets.
• Wash the protein pellet with ethanol or isopropanol, then resolubilize directly in a strong denaturing buffer such as 8 M urea, 6 M guanidine thiocyanate, or SDS-containing lysis buffer.
• Alternatively, skip TRIzol-based protein recovery and instead perform RNA extraction with TRIzol, while reserving a parallel culture for protein extraction using a RIPA or urea/SDS buffer, which often yields cleaner protein for downstream analyses.

Afzal Ahmad Maisra Azhar Thank you very much for your answers, they will be very helpful to me.