Hi!! Saranpal Chhabra

We routinely use Sarkosyl for protein solubilization in our lab. Mostly high molecular weight proteins. As you have already mentioned that your protein gets solublized in presence of sarkosyl. You only need to screen out and optimize the minimum concentration of sarkosyl which gives maximum solubility. For example you set up pilot experiment first with different concentrations of  sarkosyl (w/v) like starting from 0.05% till 0.5%. Observe SDS-PAGE and find out the minimum concentration of sarkosyl which gives maximum solubility (Here maximum doesn't mean 100% solubility, if you getting more then 60%-70% protein soluble in less percentage of sarkosyl thats better) After screening the optimized percentage of sarkosyl you set up mass culture experiment. While setting the mass culture experiment you need to calculate the sarkosyl percentage according to your pilot experiment. Its better if you take less pellets in several tubes then add sarkosyl and later pool up all the pellets after sonication and forward it for purification process..

Add the same percentage of sarkosyl in all buffers you using in purification. Its necessary because your protein is only soluble in presence of sarkosyl. After purification dialized your purified protein nicely, so all the sakosyl get removed and it can refold. Check out your protein activity with some biochemical assays and confirm it for native structure. Do not take sarkosyl percentage more then 0.3%-0.5%. take various less pellets so obviously sarkosyl percentage will also be less.

Hope this helps you. Good luck. 

Gold Biotechnology has the very useful detergent octyl glucopyranoside (OG, CAS 29836-26-8) for 1/4 the price of Sigma. The use of a removable detergent is often very important, and OG has a CMC of about 20mM it can be used to solubilize at >1% w/v and then diluted 1:10 and easily dialyzed away.  Sarkosyl can solubilize well but its very hard to get rid of in my experience.While it has similar CMC (~15mM) it binds proteins quite tightly; N-lauroylsarcosine in shampoos leaves hair silky smooth due to this durable binding.

Proving your membrane protein is in a "native like" conformation is a real challenge, good luck!

Use of Sarkosyl is not a bad idea but yes it can affect the structure and function of your protein when used in high concentration. And if you have already got a soluble protein with 0.1% Sarkosyl then why you want to go for concentrations higher that that?

Also, if you wish to use any other chemical other than Sarkosyl for solubility you can check this paper. Good luck.

 Below about 0.5% (w/v) sarkosyl is below the 15mM CMC and its usually much less solubilizing, and possibly damaging to your protein's structure.  The worst effect and the most solubilization usually occurs above the CMC. 

Hi!! Saranpal Chhabra

We routinely use Sarkosyl for protein solubilization in our lab. Mostly high molecular weight proteins. As you have already mentioned that your protein gets solublized in presence of sarkosyl. You only need to screen out and optimize the minimum concentration of sarkosyl which gives maximum solubility. For example you set up pilot experiment first with different concentrations of  sarkosyl (w/v) like starting from 0.05% till 0.5%. Observe SDS-PAGE and find out the minimum concentration of sarkosyl which gives maximum solubility (Here maximum doesn't mean 100% solubility, if you getting more then 60%-70% protein soluble in less percentage of sarkosyl thats better) After screening the optimized percentage of sarkosyl you set up mass culture experiment. While setting the mass culture experiment you need to calculate the sarkosyl percentage according to your pilot experiment. Its better if you take less pellets in several tubes then add sarkosyl and later pool up all the pellets after sonication and forward it for purification process..

Add the same percentage of sarkosyl in all buffers you using in purification. Its necessary because your protein is only soluble in presence of sarkosyl. After purification dialized your purified protein nicely, so all the sakosyl get removed and it can refold. Check out your protein activity with some biochemical assays and confirm it for native structure. Do not take sarkosyl percentage more then 0.3%-0.5%. take various less pellets so obviously sarkosyl percentage will also be less.

Hope this helps you. Good luck. 

Hi Gaurav!

Reading your question and some answers to it, i could find out that the solubilizing agent being used for the current study has extremely high cmc. 15 mM is extremely high concentratiob. Why don't you use some other bio-compatible agent having low cmc values. Will it useful for the study??

Hi,

I routinely use 2% sarkosyl during the step of sonication of E.coli cells ... and the activity of my proteins is not influenced. 

Sarkosyl is much more mild than SDS.

Hello Saranpal,

You can try physical methods, let say, circular dichroism and differential calorimetry scanning to watch conformation changes as native, so denatured protein. 

Good luck!

Yuri

Hi,

I would like to add my query as well here in this forum.

One of the proteins that I am looking to study is intrinsically in aggregated form in the cell even in normal conditions. Hence, getting a sds- page and western blot for the protein is tough. The cells are animal tissue culture cells.

Can some one guide me with detailed protocol as how to make this protein soluble to be noticed on the western blots.

Thanks in anticipation.

Cheers,

Apurva Agrawal

What is the incubation time and temperature you guys used for sarkosyl?

Hello, I would like to add some questions to this one.

I have successfully cleaved by protein of interest from it's GST-tag using PreScission protease (validated by coomassie), following purification using glutathione sepharose. However I cannot elute my protein! It remains firmly bound to the glutathione sepharose beads.

At the beginning, to increase solubility of the protein I added sarkosyl 1%, triton 2% and chaps20 mM to the lysis buffer, and it seemed ok, afterwards the lysate was incubated with glutathione sepharose beads overnight, washed with PBS1x, and eluted in Tris50mM pH 7-8.0, NaCl 150mM, DTT 1 mM with the protease. The cutting was ok but the protein cleaved remains with the beads, I added Triton or CHAPS to the elution buffer but without result, the protein still remains on the beads fraction. Should I need to eluate with another detergent?

Could any one bring me a solution?.

Thanks 

Rosa, I think your protein has merely become insoluble on the glutathione resin, after you cut off its solubility tag. The glutathione binding tag eluted off?

You may want to screen elution conditions, and a handy way to do this is to divide the resin into multiple spin columns and follow elution by A280nm or SDS-PAGE. A simple frit to retain the resin works well and lets you see any soluble eluant but a variation could be to use a MWCO spin column which would only show your protein if its a monomer. Don't be afraid to use strongly denaturing conditions in a couple of samples (chaotropes like urea, GuHCl) as these may be required to solubilize. If denaturation is required for solubility then doing another screen for soluble refolding (with MWCO filters) has worked very well for me in the past. Good luck!

Thank very much, I will try.

Rosa, not sure if the GST-protein is still an issue, but there is a paper that pertains directly to your problem with sarkosyl alone. See Anal. Biochem. (1993) vol 210, pp179-187. They had the same issue with the protein binding to the beads.