Not sure if U have what you need, but you might try cetylpyridium chloride (CPC) to ppt the polysaccharides.. I am semi retired life science professional and haven't used this technique in at least 30 yrs, but I recall that it can remove acidic polysaccharides from a sample treated w/ protease and then TCA. I am proposing to use CPC 1st. .

Cant you use cold acetone? After precipitation and centrifugation you can remove the excess acetone and evaporate the rest, only the proteins remain

Would cold acetone not precipitate polysaccharides?

Cold acetone would be the best option but still it needs to be tested with your sample first!!! Good luck

Dear Iqbal, I dont think so, it is a classical mathod to precipitate protein. Note, please that not always it is possible to recover proteins with full activity often not even into the original solution. If youre testing enzymes, many might be killed by precipitation. Another option would be molecular filters like Centricon, however this would depend on your knowing the sizes of molecules in your sample in order to choose filters. This method on the other hand, is much lesss agressive to enzymes.

Dear Fox, i did agree with you but we can give it a initial trial. since the other methods like one you suggested need prior conformational information of the molecule understudy.

Many Thanks

Haroon

I propose ammonium sulphate precipitation. It's quick and easy. , I recomend grinding finely the salt and adding gently to an ice-cold solution of your sample. Try different concentrations, centrifugate the samples and resuspend the protein pellets with a suitable buffer. Here you have a calculator

http://www.encorbio.com/protocols/AM-SO4.htm

Not sure if U have what you need, but you might try cetylpyridium chloride (CPC) to ppt the polysaccharides.. I am semi retired life science professional and haven't used this technique in at least 30 yrs, but I recall that it can remove acidic polysaccharides from a sample treated w/ protease and then TCA. I am proposing to use CPC 1st. .

This is trully interesting Alan.

Eduardo's method worked quite well, I think. I have now managed to fractionate the water extract to 3 clearly visibly different looking fractions, a dried supernatant, polysaccharides (by EtOH precipitation, and than proteins with cold acetone precipitation.

Many Thanks Every One, esp Eduardo.

Haroon

The purpose of this work is to use the extracts with cells, as part of an assay, and although cetylpyridium chloride is a really good suggestion and is without doubt appealing as it remains solid up to 77degC and insoluble in acetone, my concern is trace amounts which may be toxic to cells, that is why I decided not to go with the TCA route.

Have you tried ammonium sulfate precipitation. It works best to precipitate protein

will I be able to remove the salt after, effectively ?

Certainly, by re suspend your ammonium sulfate pellet in a low salt buffer. Also polysaccharide can be precipitated and removed from sample using 0.1% cetavlon

Also, you can ensure with dialysis in a suitable buffer, overnight at 4 ° C

Sound like you want to preserve the protein function after precipitation. I have used organics like acetone or acids but resuspension is difficult.

Pehaps you should consider a lectin-sepharose approach or somthing similar to remove the polysaccarides first before processing your sample.

Good luck with your research.

If your protein isn't glycosylated itself, you might be able to use a borate column to selectively bind and remove the carbohydrates. Sigma and Bio-Rad sell resins with borate-containing compounds which selectively bind molecules with adjacent (cis-diol) hydroxyl groups like carbohydrates.

If you don't need the proteins then you could precipitate them (the proteins) by adding 9 volumes of ice cold acetone. Mix the solution and set is on ice for 30 minutes. Centrifuge to remove the precipitated proteins.

Prof Richard, I also want to precipitate the residual protein to take out sulfated glycosaminoglycan (GAG) from protease digested crude GAG sample. I use protease to digest all the protein in tissue sample but It couldn't work properly as after centrifugation, I precipitated the supernatant with ethanol and then collected the precipitate by centrifugation, I found that still there is plenty of protein left in my sample. Now I want to take out that protein and after that I will take out DNA, to get a good fraction of sulfate glycosaminoglycan, can you suggest me something.

If I use Cold acetone, will my GAG will be suspended in supernatant or it will also go in precipitate with protein.