Just two days ago I posted a question here regarding the protein extraction with TriZol - https://www.researchgate.net/post/Protein_isolation_with_TriZol_TriPure-huge_white_pellet_after_the_final_centrifugation

Although many say that it's "not working" and the pellet doesn't dissolve, I could say that from the experience I had, the pellet does dissolve quite easy at 50C in 1% SDS for 15 min if:

- the phenol phase remained after the aqueous phase removal is processed up to the point of storage the pellet in GuHCL/EtOH and is processed in parallel with the aqueous phase, e.g. two people are needed to continue from the moment the phases are separated. It's tempting to freeze the phenol phase and process it later, but I believe that's no good for the final pellet dissolving.

- after the first wash in GuHCL, the pellet should be broken in as many pieces as possible (by the tip)

- the protein pellet is not too big. How much is "not too big" is questionable though.

For me the major challenge occurs after the solubilization of the pellet (as written in my other post). I already sent emails to Roche and Invitrogen asking for help.

On another side, I personally would prefer to work with a phenol-free, RNA+protein kit. Currently I'm waiting for a sample from the MN Nucleospin RNA/Protein kit.

I had the same idea a couple of years ago... did not like it one bit. The protein pellet obtained with the TRIzol kit is very tricky to resuspend... gummy and disgusting. I had been used to resuspend proteins precipitated with acetone, metaphosphoric or trichloroacetic acid. This pellet, however, gave a fight. I did not have a lot of time on those days so I did not follow up on that. My best results were obtained with a pellet pestle adjusted to a manual motor, but resuspension was far from quantitative. Sorry I can't give you more details! Best wishes!

Just two days ago I posted a question here regarding the protein extraction with TriZol - https://www.researchgate.net/post/Protein_isolation_with_TriZol_TriPure-huge_white_pellet_after_the_final_centrifugation

Although many say that it's "not working" and the pellet doesn't dissolve, I could say that from the experience I had, the pellet does dissolve quite easy at 50C in 1% SDS for 15 min if:

- the phenol phase remained after the aqueous phase removal is processed up to the point of storage the pellet in GuHCL/EtOH and is processed in parallel with the aqueous phase, e.g. two people are needed to continue from the moment the phases are separated. It's tempting to freeze the phenol phase and process it later, but I believe that's no good for the final pellet dissolving.

- after the first wash in GuHCL, the pellet should be broken in as many pieces as possible (by the tip)

- the protein pellet is not too big. How much is "not too big" is questionable though.

For me the major challenge occurs after the solubilization of the pellet (as written in my other post). I already sent emails to Roche and Invitrogen asking for help.

On another side, I personally would prefer to work with a phenol-free, RNA+protein kit. Currently I'm waiting for a sample from the MN Nucleospin RNA/Protein kit.

My experience for protein extraction using trizol reagent was very bad... however, Petar suggestion ("pellet does dissolve quite easy at 50C in 1% SDS for 15 min") seems to be a ray of light... Shall try it... thanks Petar...

I have used it at 50C for sometime and could not get dissolved protein. well yes storage for overnight might have hardened the pellet. I will try with fresh processing and no storage. Ya RNA plus protein kit should be helpful.

U could also try Urea or urea + SDS. THat gave us better results while isolating total protein from human blood cells with TRI R.

Hi deepak, If urea them whats the conc to be used? I mean final conc in the solution? and same question for SDS, what % ?

8% U and upto 1% SDS. Go thru this article for more detailed info. Ur problem gets solved with confidence. Study well before and during work and ull nyoy

it !!.

http://www.biotechniques.com/BiotechniquesJournal/2007/April/Isolation-and-solubilization-of-proteins-after-TRIzol-extraction-of-RNA-and-DNA-from-patient-material-following-prolonged-storage/biotechniques-41641.html?pageNum=3

Thank you for the suggestion Deepak. I do study for the experiments I perform. This query is an indirect one from one of my colleagues who is an MD and doesnot work much with mol. Bio.

Glad to have the link.

Hi Peter,

I also got a hard time solubilizing my protein pellet using Trizol protocol.

I don't get it when you say: "

the pellet does dissolve quite easy at 50C in 1% SDS for 15 min if:

- the phenol phase remained after the aqueous phase removal is processed up to the point of storage the pellet in GuHCL/EtOH"

What deviation are you doing from this standard procedure:

1. Following RNA extraction procedure using Trizol until you remove supernantant aqueous phase. Add EtOH 100% (300 uL) to the bottom phase, mix by inversion and let stand 2-3 min at r.t. Spin (4500 rpm, 4C, 5 min).

2. Remove phenol/ethanol supernatant (the pellet contain DNA and can be further process) and precipitate proteins by adding iPrOH (1.0 mL). Let stand 10 min, r.t. Spin 13000 rpm, 10 min , 4C)

3. Remove supernatant and add 0.3M guanidine hydrochloride in 95% EtOH (1 mL). Disrupt the pellet from the bottom. Let it stand 20 min, r.t. Spin (10 000 rpm, 5 min, 4C). Repeat twice.

4. Remove supernatant and vortex protein pellet in EtOH 100% (1 mL). Let it stand 20 min, r.t. Spin (10 000 rpm, 5 min, 4C).

5. Remove the EtOH and air dry 5-10 min. Dissolve in SDS 1% (100 uL) by pipetting. Incubate (50C, 10 min). Spin (13000 rpm, 10 min, 4C) and transfer the supernatant to a clean tube.

many thanks for your help!

I just tried

2% SDS and 3% B-mercaptoethanol  in 50 mM Tris (pH 8.8) 

the pellet completely redissolved in 100-200 uL, although less may even work.

Thanks Brendan. will try.

I do that in routine, you need just a specific suspension buffer. Urea + Chaotropic agents. It also works for mass spectrometry if the urea is not too concentrated (below 3M). Tell me if you interested, i could share the suspension buffer i use.

Cheers !

Hello

i agree with all . and links here will help you

Good luck

http://www.sciencedirect.com/science/article/pii/S0003269716300860

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211643/

Well i agree with everyone, protein extraction using trizol is very hard. Since its difficult to dissolve the pellet.

I had a very small sample, L3-L5 spinal cord segment, obtained from mice. from the segment,Using trizol i extracted RNA. then protein as per the protocol. Just after the first washing step, the pellet was gummy and could no longer dissolve, using the tip of a pipette i made small pieces, followed by brief sonification. The pellet did not dissolved completely, Untill the end of all the required steps. I centrifuged as required. took the supernatant, and discarded the undisolved pellet. Measured the protein concentration using BCA reagent, and yes it was enough for my further experiments.