Extracting protein from tissue samples and cultured cells is Step #1 in many biochemical and analytical techniques. Before you can do a Polyacrylamide Gel Electrophoresis (PAGE), a Western blot, or mass spectrometry you need to extract your protein. Nowadays, a lot of labs have switched to kits for their protein extraction but these kits can be more expensive and can have poorer protein yields than the tried and tested method I will talk about today…The TRIZOL® method!
Now, you may have heard that TRIZOL® extraction can be a little fussy especially for beginners. But don’t fear it really is not all that bad. And getting amazing results from both cells and tissues, fresh and frozen samples are worth some careful pipetting. Plus this technique can also be used to extract DNA and RNA!
A Brief Introduction
Before we get down to business let me tell you about the important players in this method:
- TRIZOL® a.k.a. TRI reagent, as it was called by it’s creators, solubilizes biological materials and denatures protein. It is used to disrupt your cells and dissolves their cellular components.
- Chloroform. After centrifugation, chloroform separates the TRIZOL® solution into two phases: the aqeuous phase and the organic phase. The aqueous phase will contain your RNA, which you can precipitate later by isopropyl alcohol. The organic phase will contain your DNA and protein.
- Ethanol. Once you’ve siphoned off the aqueous phase, you can use ethanol to recover your DNA from the organic phase.
- Isopropyl alcohol. Once you have removed the DNA you can perform an additional precipitation step using isopropyl alcohol to isolate your proteins.
Getting Down To Business
If you’re using cells and are hoping to quantify the amount of protein present, you need to start with a known quantity of cells. If you are using tissue, either fresh samples or samples frozen in liquid nitrogen the procedure is the same as with cultured cells. Only difference is a pellet of cultured cells is simply resuspended in TRIZOL® while tissue samples needs to be actively homogenized. And remember, if frozen, allow your tissue time to melt before homogenizing.
Clean your fume hood with 70% ethanol.
If you are processing a tissue samples use tweezers to add approximately 1mm3 of your tissue sample to 1 ml of ice-cold TRIZOL® and determine the weight on your sample. If your sample is fresh keep it on ice. If your sample is frozen give it 5 minutes to thaw at room temperature before putting it on ice. Let your sample sit on ice for 5 minutes.
If you are processing cultured cell samples add your desired number of cells in media to a 1.5 ml eppendorf tube and centrifuge at 300g for 5 minutes. This will pellet your cells. Next aspirate the media and resuspend the cells in 1 ml of ice-cold TRIZOL® (5-10 x 106 cells/1 ml) and leave to sit on ice for 5 minutes.
Add 200 µl of chloroform for every 1 ml of TRIZOL® and shake your samples vigorously for 15 seconds, and leave at RT for 3 minutes. Spin at 12,000g for 15 minutes at 4°C. This will sepate your aqueous and organic layers.
Aspirate the clear upper aqueous layer (approximately 700 µl for tissue and 400 µl for cells). This layer contains your RNA. Once gone, add 0.3 ml of 100% ethanol per 1 ml of TRIZOL® to the remaining organic layer. Mix samples by repeated inversion and leave at room temperature for 2-3 minutes.
Next sediment your DNA by centrifugation at 2,000g for 5 minutes at approximately 4°C. Then precipitate your proteins (approximate volume 0.8 ml per 1 ml TRIZOL®) with 1.5 ml of isopropanol per 1 ml TRIZOL®. Leave samples for 10 minutes at room temperature.
Centrifuge your sample at 12,000g for 10 minutes at approximately 4°C. Remove the supernatant and wash the protein pellet thrice in a solution of 0.3 M guanidine hydrochloride in 95% ethanol.
Add 2 ml of guanidine hydrochloride wash solution per 1 ml of TRIZOL® Reagent used for the initial homogenization. During each wash cycle, store the protein pellet in the wash solution for 20 minutes at room temperature and then centrifuge at 7,500g for 5 minutes at approximately 4°C. Vortex the protein pellet in 2 ml of ethanol and leave to sit for 20 minutes at room temperature.
Centrifuge at 7,500g for 5 minutes at approximately 4°C. Remove supernatant, leave pellet. Let pellet air dry for no more than 10 minutes. Dissolve it in 1% SDS by pipetting. Complete dissolution of the protein pellet may require incubating the sample at 50°C.
Centrifuge at 10,000g for 10 minutes at approximately 4°C, and transfer the supernatant to a fresh tube. The sample is ready for use or may be stored at -20°C.
For more details on the amount of protein you can expect for different sample sizes and on the DNA and RNA isolation and solubilization, see here.
Tips, Troubleshooting, and Tricks
- If you’d like to keep your RNA intact, clean your hood with RNase ZAP as well as ethanol before you start. For that matter you should also clean all your pipettes and any holders, tip boxes etc. with ethanol and RNaseZAP.
- If you use 1.5 ml eppendorf tubes, it can be very difficult to homogenize the tissue adequately and in a good amount of time. You may want to perform the homogenizing step in a larger conical tube.
- To determine the weight of a tissue sample, pre-weigh your 2 ml eppendorf containing the TRIZOL®. Re-weight once you’ve added your sample. Subtract your answer from the protein-free weight of the tube.
- After you’ve added your TRIZOL® to either cell or tissue samples they can be stored at -80°C. You can then process your sample later.
- Whenever possible keep your sample on ice.
- Overdrying your protein pellet is disastrous! If your sample isn’t dry after 10 minutes, move on. To help it dry, I like to leave the tube inverted on kitchen paper but at an angle so the fluid forms a drop and runs down the side. You can also take a tiny sliver of paper and dab the rim to help it move. Don’t tap the tube too hard, your pellet may detach! The goal is to remove any excess water but not dry out your pellet.