13 replies to this topic

[Negar]

Hi all!

I cannot get rid of Polysorbate 80 (Tween 80) from my protein solution. Neither dialysis nor ultrafiltration (using Amicon ultra filters) worked. Any suggestions? plz note that I need the intact protein at the end.

[mdfenko]

if neither dialysis nor diafiltration worked then you may want to try precipitating the protein away from the tween solution with ammonium sulfate or acetone (first check to make sure that tween 80 remains soluble under those conditions).

[Negar]

mdfenko, on 23 February 2011 - 08:40 AM, said:

if neither dialysis nor diafiltration worked then you may want to try precipitating the protein away from the tween solution with ammonium sulfate or acetone (first check to make sure that tween 80 remains soluble under those conditions).

Previously I had tried cold acetone ppt (at -20 C) and I repeated it again after your post. My protein does not go back to solution-phase after precipitation.

[mdfenko]

are you sure that your protein is soluble? the tween may have been necessary to solubilize the protein

it's possible that your protein denatured in the acetone. you could try fractional precipitation with acetone, it may be more gentle or try ammonium sulfate precipitation.

[Negar]

mdfenko, on 28 February 2011 - 11:54 AM, said:

are you sure that your protein is soluble? the tween may have been necessary to solubilize the protein

it's possible that your protein denatured in the acetone. you could try fractional precipitation with acetone, it may be more gentle or try ammonium sulfate precipitation.

[Negar]

my protein is hydrophobic so to some extent tween is needed.
I am not quite sure what do you mean by "fractional precipitation with acetone". I used the protocol from Pierce:

http://www.molecular...3-1/G3-1-17.pdf

[mdfenko]

a long time ago i used to do a fractional precipitation with acetone. i would precipitate some contaminating proteins with 40% acetone then precipitate my protein of interest by raising the acetone to 50% (centrifuging in between, of course). the procedure from pierce is to precipitate all of the proteins present.

you can do a buffer exchange by gel filtration then concentrate the protein.

if the tween is necessary then you may want to reconsider its removal. why do you need to remove it?

[Negar]

mdfenko, on 28 February 2011 - 12:41 PM, said:

a long time ago i used to do a fractional precipitation with acetone. i would precipitate some contaminating proteins with 40% acetone then precipitate my protein of interest by raising the acetone to 50% (centrifuging in between, of course). the procedure from pierce is to precipitate all of the proteins present.

you can do a buffer exchange by gel filtration then concentrate the protein.

if the tween is necessary then you may want to reconsider its removal. why do you need to remove it?

yes. i need to remove it bcs the next step for me is ESI-MS. Tween suppresses the protein signal and also is a contamination for my experiments.

[mdfenko]

in what medium do you need the protein?

as i mentioned in the last post, you may be able to exchange the buffer by gel filtration but you have to exchange it to something in which the protein will remain soluble and is compatible with esi-ms (and won't hurt the gel filtration matrix).

[Negar]

mdfenko, on 28 February 2011 - 01:13 PM, said:

in what medium do you need the protein?

as i mentioned in the last post, you may be able to exchange the buffer by gel filtration but you have to exchange it to something in which the protein will remain soluble and is compatible with esi-ms (and won't hurt the gel filtration matrix).

Ammonium acetate would be a good buffer for my protein.

I was thinking about exchanging tween 80 with some other detergents using dialysis. Could you suggest a detergent(s) to be replaced with tween 80?  I do not know any MS-compatible detergents  

[mdfenko]

i'm attaching a pdf of a mass spec handbook, from g-biosciences, which includes some information on compatible materials.

it mentions triton x-100 and nonidet p-40.

they also have a detergents handbook available at their website (it's too big to attach).

Attached Files

•  mass spec handbook.pdf   2.63MB   217 downloads

[Negar]

Thanks sooo much!!! I'll take a look.

[proteaMatt]

I often prepare samples for analysis with ESI or (LC)MALDI-TOF. Something that I use, especially when I want to lyse cells, is an acid labile surfactant. These are really effective for solubilization of proteins with an ammonium bicarbonate buffer. The beauty of a reagent like this is how easy it is to inactivate it in your sample. Usually I add enough TFA or Formic acid to lower the pH below 3 and the surfactant will degrade. Another chemical you might also consider using in conjunction with this would be a small amount of NDSB 201 which can help to solubilize proteins as well.  

[Negar]

Thanks for your help! I guess my protein will be denatured at pH=3. I need to get the intact protein at the end.