Bio-beads are often used to remove Triton X-100.

Dear Sai Supriya,

one possibility is to make first an exchange of the detergent and select one for the exchange that can be better removed by dialysis than triton x . One i know is Octyl β-D-glucopyranoside. It  has many advantages, one is that it can be easily removed from the protein solution.

Bio-beads are often used to remove Triton X-100.

Hi there,

If your protein is tagged, you can use affinity column to immobilize it first and then exchange buffer for a triton free one. If not tagged, the same operation can be done using suitable IEX column.

Triton X-100 gives high absorbance at 280nm. For Triton removal I would suggest to attach your protein to the resin (e.g. NiNTA) and wash it with buffer without Triton. Or you can use Bio-Beads as Adam suggested.

What kind of protein do you purify? Is detergent necessary for its stability?

Another way  to measure concentrations is BCA assay, of course if you don't have any reducing agents in your buffer. Bradford is probably not going to work because of high concentration of detergent.

As said , Dialysis for 2 days with repeated change of buffer for every 2 to 3 hours will do the trick , simple and not costly.

hi everyone, now I'm purifying my protein chitin beads, but I can not elute it down,

 so can I try Triton X-100? and which concentration? thx

Is the Triton X-100 actually needed to elute the protein from the beads, or is it there to keep the protein in solution once it is eluted? If it is the latter case, you may be able to use a much lower Triton concentration to keep the protein in solution. The CMC is about 0.013%, so you might be able to use 0.1% or less.

@ Adam as you said CMC is about 0.013% so using 0.1%  is above CMC. Isn't it? Also if I my rpotein sample has 0.5% troton X 100 will it entrap protein in the micelles? Will it affect the assay?

Please suggest.

Thank you

Hello Pratik!

True that, sometimes. Your protein could look stable in a buffer with 0.5% Triton X-100. Reality "could" be that, it is not feeling the buffer conditions around being entrapped in the micelle. Many assay kit makers warn the interference of ionic/non-ionic detergent interference, so, probably yes.

Hope this helps.

Good luck!

Hello Tengfei Liu!

You can try using elution buffer with pH extremes like 4 or 10 until it does no harm to your protein. Adding detergent too does help, within its CMC (critical micellar concentration).

Hope this helps.

Good luck!

I am doing ITC in a buffer supplemented with 0.5% triton x100 and I see Nucleotide binding response. (when I don't use triton x100 protein tend to precipitate). Now according to the binding curve it doesn't  seem that protein is getting entrapped in the micelle. (N value is nearly 1 meaning correct molar ratio of protein and nucleotide)

Also, If considering detergent CMC is so critical (which off-course should be) why is it then some of the NEB reaction buffers have got 0.025% to 0.1% tritonx100, which is more than that of CMC of triton x100. 

see the links below-

https://www.neb.com/products/e1201-quick-blunting-kit

https://www.neb.com/products/m0267-taq-dna-polymerase-with-thermopol-buffer

Thank you

Dear Supriya,

I have been using Ionic detergent compatibility reagent to exclude the detergent interference while measuring the protein cocn. I have used this, it gives a reliable OD value to some extent. You might try this, hope it helps.

Best wishes.

@saraswathi subramanian, Could you tell me what this ionic detergent reagent could be? Is it commercially available or has a recipe to prepare in the lab?

I was using a commercially available one. Just use the link inhere  https://www.lifetechnologies.com/order/catalog/product/22663  and unfortunately I didn't find any composition to prepare by our own.