Amruta

the answer for your question is No.. you dont need to refold protein.

Triton -x -100 is a classical example of a non-ionic detergent. The nonionic detergents differ with the ionic ones in that their headgroup is uncharged and hydrophilic. They are considered mild surfactants as they break protein-lipid, lipid-lipid but not protein-protein interactions and most of them do not denature proteins. Therefore, proteins are solubilized and isolated in their native and active form retaining their protein interactors. This is their main advantage and preferred in isolation of membrane proteins. Triton X-100 is a non-ionic detergent used

to denature cell membranes without denaturing the protein. Triton X-100 breaks up protein aggregates to promote enzymatic activity.

information sourced and complied from multiple papers / vendors

I assume your protein is in inclusionbodies and you did not use denaturants such as Urea or Guanidine HCl. Triton-X100 can denature proteins. It is typically used to clean up inclusion bodies (get rid of membrane proteins etc.). Regardless I would remove Triton-X-100 by dialysis or ionexchange. Remember that complete removal of Triton is very difficult. If possible I would use alternate detergent with smaller size.

http://www.piercenet.com/browse.cfm?fldID=5558F7E4-5056-8A76-4E55-4F3977738B63

Good luck

Kumar

It depends. How much of Trion X-100 is used in your sample preparation?

As you mentioned, it is not an ionic detergent like SDS, so the structure of protein might still be preserved. I dont think Nondenaturing detergent such as Trion X-100 can completely UNFOLD the protein structure as Urea/GdnHCl does. May be you don't have to refold it. they generally do not disrupt native interactions and structures of water-soluble proteins and do not have cooperative binding properties. The main effect of nondenaturing detergents is to associate with hydrophobic parts of membrane proteins, thereby conferring miscibility to them.

If you are going for structure studies (NMR/crystallography) Trion X-100 is not generally desirable. I would try to remove Trion X-100in my buffer/sample by ion exchange chromatography(size exclusion wont help) and dialysis may be.

You may want to have a look at these pages:

http://www.piercenet.com/browse.cfm?fldID=5558F7E4-5056-8A76-4E55-4F3977738B63

http://www.labome.com/method/Detergents-Triton-X-100-Tween-20-and-More.html

If you are expecting a non-denatured protein after the process, I suggest you'd better to test it by some kind of spectroscopy methods. FTIR or CD spectroscopy can give an idea about protein denaturation from their spectra and their secondary structure analysis. There are numbers of examples in the literature related with protein secondary structure alterations monitored by CD or FTIR spectroscopy. So, if possible, it is better to perform a spectroscopic analysis to be sure.

Hi, I am using Yeast expression system. Hence, there are no inclusion bodies here. My protein gets expressed in the pellet of the yeast culture after 13000rpm spin. I solubilize the pellet to extract the protein. I can use Urea but it will denature the protein. That is the reason i used detergent. I want to know whether I should refold my protein or not even though it is under non de- naturing conditions?

Triton X-100 is not supposed to denature proteins if you use it at reasonable concentrations (up to 1-2%). At higher concentration, denaturation may be possible depending on your protein (I don't believe you can predict it). Since renaturation can be a tedious and inefficient process, I would advise you to purify the solubilized protein in its current state, and check if the protein is "correctly" folded using any functional or structural assay (binding to a natural ligand, enzymatic activity, if available: comparison of NMR or circular dichroism spectra with that of the native protein).

Good luck!

do you have biotinylated ligand..? can you do some plate based or SPR based ligand binding studies..? if it binds the liagnd your questions is answered. Triton X-100 should not denature the protein. get rid of the detergent using pierce detergent removal spin columns. its a 5 minutes protocol. but the question is after removal of the detergent will your protein be folded or beocme insoluble and unfold..? well you have to try a small portion of the protein and see solubility/ligand binding to answer that... then you might have to employ ion-exchnage/dialysis etc to slowly remove the detergent. you can try arginine buffers while removing detrgents to keep protein solubilised.

I have nothing new to add, but I agree strongly with Ludovic Cartier that you should purify the solubilised protein in its current state and use all possible means to determine whether or not it has retained its "native" state.

One cannot generalise in such matters. You have to establish the state of a protein after Triton X-100 treatment in each case.

I forgot to mention that I often use 0.5% Triton X-100 in my lysis buffers to improve cell lysis. Using a Ni-NTA column (IMAC) for purification, I've never encountered problems for removing the detergent. Once the protein bound to the column, washing extensively with the equilibration buffer before elution is generally enough.

To monitor/optimize protein purification conditions, it is always very beneficial to have some kind of assay (e.g., activity, analytical/biophysical methods). Best luck.

it would depend on what you are using your protein for- would triton interfere with the assays/ measurements you are using it for?If they might, I would recommend trying to adapt your purification protocol though so that no addition of triton is needed . If you are refolding- do it slowly by dialysis or similar but my experience is the same as Balananda's that complete removal is a complete nightmare. Good luck !

I am agree with Ludovic. Chances of getting good refolding are slim. You should be more specific about what protein are you working with, as your results may be completely different for a membrane or a cytoplasmic protein. In general people use Triton X100, Tween 20 and NP40 in this order as detergent with different (decrease) stringency to affect proteins structure, although is just a general rule (not sure if was checked experimentally).

Amruta

the answer for your question is No.. you dont need to refold protein.

Triton -x -100 is a classical example of a non-ionic detergent. The nonionic detergents differ with the ionic ones in that their headgroup is uncharged and hydrophilic. They are considered mild surfactants as they break protein-lipid, lipid-lipid but not protein-protein interactions and most of them do not denature proteins. Therefore, proteins are solubilized and isolated in their native and active form retaining their protein interactors. This is their main advantage and preferred in isolation of membrane proteins. Triton X-100 is a non-ionic detergent used

to denature cell membranes without denaturing the protein. Triton X-100 breaks up protein aggregates to promote enzymatic activity.

information sourced and complied from multiple papers / vendors

In addition to the fact that Triton doesnt denature ur protein, the His-tag tail should be in exposed position for binding to NTA-resin. Regardless your protein is fold or no, as long is the is freely exposed, then you dont have to be worry (if your concern is only during purifcation step). In fact, I had no problem using 1% Triton containing buffer during Ni-NTa purification without refolding step.

Triton never disturbs protein in initial stage separation , But still check out what pH you have maintained. Some times due to close values of iso-electric point may affects.

Above points are good. Does the purified protein bind the NTA-resin? That would tell you if triton x-100 created problems. Have you tried binding the purified protein with the core sequences (short peptides?) of your ligand ? Do you have an assay to detect binding? The binding may require certain new buffer constituents, and its good to check if your pure protein is stable under these conditions. Hope this helps..Good Luck!

Attached articles could answer your question.

Good luck