Ammonium sulfate precipitation from solutions with high sugar content

[WS]

Dear Experts,

I am attempting to precipitate small amounts of (total) protein from a
fruit juice concentrate. It looks quite much like honey. My plan is to
dilute it 1x with water (to reduce viscosity) and then saturate it
with ammonium sulfate. As the solution contains high amounts of sugars
(mostly saccharose, I assume), will these interfere with my
precipitation process?

If yes, what may I do to circumvent it? Probably dialysis, but any
other ideas?

Thanks for your help!

Wo

[Pow Joshi]

Hi Wo,

I was wondering if you could simply use the crude extract to spin down and
create a sucrose density gradient...that will allow you to pick out some
proteins within the gradient, and then the remaining may go over for
ammonium sulphate precipitation ? ...I've no idea how this would work, but
just a thought, if you have'nt already considered it.

Pow

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[WS]

Hi Pow, long time no see :)

My big problem is that there is almost no protein in the sample -> I won't see any bands. I expect about 1mg from 50 ml. I have started with 50ml of fruit juice concentrate, diluted with 50ml water and then added ammonium sulfate until saturation: No visible turbidity, No (visible) pellet after 2hrs @ 4000g (except some ammonium sulfate crystals. Unfortunately, I do not have access to a high speed centrifuge for such volumes. Spinning 24 eppis with 2ml each @ 15.000g in my small benchtop centrifuge might be an option, however.

Could it make sense to add some unrelated protein, eg BSA or OVA (that's what I have in my fridge), as co-precipitant (like one does with glycogen or poly-acrylamide for DNA)?.

Wo

[Michael Sullivan]

What do you want to use the protein for?

I had a very dilute, yet viscous extract of flowers that I wanted to use for western blotting. Carbohydrates made it visous, interfered with protein assays, and made the samples run quite poorly in a gel. I ended up using a phenol extraction method followed by MeOH precipitation that worked quite nicely. Of course, if you are trying to recover active protein this could be a problem.

Mike
---
Michael L. Sullivan, PhD
Research Molecular Geneticist
US Dairy Forage Research Center
1925 Linden Drive
Madison, WI 53706
608-890-0046 (Phone)
608-890-0076 (FAX)

[WS]

[WS]

Hi Michael, I do not need active protein, I need it for a western. I'll give it a try, thanks!

Wo

[Irit Rappley]

Hi Wo,

In the past I've added 1 mg/mL BSA to a dilute protein solution, then precipitated it with TCA. However, then you'll be stuck with 1 mg/mL BSA in your resulting pellet.

If you're seriously considering the 24 eppis option, wouldn't dialysis be easier?

HTH,
Irit

_______________________________________
From: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] On Behalf Of WS [novalid...@nurfuerspam.de]
Sent: Monday, April 30, 2012 2:22 PM
To: bionet.molbio....@googlegroups.com
Cc: met...@magpie.bio.indiana.edu; WS
Subject: Re: Ammonium sulfate precipitation from solutions with high sugar content

[WS]

[Pow Joshi]

well, here's a plant phloem/Xylem sap protein isolation I found....
http://www.springerlink.com/content/h8622j4747246137/#section=82175&page=4&locus=23

They use Centricon ultra filtration step to remove the sugars it seems to
me .... followed by TCA/acetone precipitation ....

[Pow Joshi]

and here's one more, from sugarcane :)
http://onlinelibrary.wiley.com/doi/10.1002/elps.200900779/pdf
on quick view it seems to me that they follow the method that Michael
suggested, however, they also seem to have comparative yields from
different methods ... hope that helps
Pow

On 1 May 2012 11:52, Pow Joshi <pow....@gmail.com> wrote:

> well, here's a plant phloem/Xylem sap protein isolation I found....
> http://www.springerlink.com/content/h8622j4747246137/#section=82175&page=4&locus=23
>
> They use Centricon ultra filtration step to remove the sugars it seems to
> me .... followed by TCA/acetone precipitation ....
>
>
> On 1 May 2012 07:18, WS <novalid...@nurfuerspam.de> wrote:
>

[Dr Engelbert Buxbaum]

In article <mailman.239.13358...@net.bio.net>,
novalid...@nurfuerspam.de says...

>
> Hi Pow, long time no see :)
>
> My big problem is that there is almost no protein in the sample ->

Iwon't see any bands. I expect about 1mg from 50 ml. I have started with

50ml of fruit juice concentrate, diluted with 50ml water and then added
ammonium sulfate until saturation: No visible turbidity, No (visible)
pellet after 2hrs @ 4000g (except some ammonium sulfate crystals.
Unfortunately, I do not have access to a high speed centrifuge for such
volumes. Spinning 24 eppis with 2ml each @ 15.000g in my small benchtop
centrifuge might be an option, however.
>
> Could it make sense to add some unrelated protein, eg BSA or OVA
> (that's what I have in my fridge), as co-precipitant (like one does
> with glycogen or poly-acrylamide for DNA)?.
>
> Wo

Hi,

what Wo presumably was referring to was a self-forming gradient (similar
to CsCl). This can be done, but requires an ultracentrifuge.

In your situation, I would try either dialysis against high MW PEG or an
Amicon pressure cell to remove most of the sugar and fluid. You could
even dilute the product of such an operation with buffer and repeat.