polysaccharide purification - (Jul/15/2008 )

hi


any idea of bacterial polysaccharide purification.
want to remove DNA and proteins.

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See these links, let us know what worked!
http://search.vadlo.com/b/q?sn=158621799&a...ation&rel=0
..

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the articles in the above link focus on purification of DNA and RNA, whereas my concern is totally opposite.
I tried to use these methods the other way round but satisfactory results were not obtained.
i konw that PS purification involves some sort of ethanol extractions but percentage m not sure of.
SO needed help regarding that.

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May be then you can check these links!
http://search.vadlo.com/b/q?sn=158621799&a...k=LPS&rel=0
..
Somebody else need to jump in if I am again way off!

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Here's my protocol:

1. Inoculate 10 ml of appropriate media with growth from an agar plate, and incubate overnight.
2. Transfer the 10 ml overnight culture to a 100 ml bottle of fresh media, and allow to grow for ~24 hours.
3. Transfer the 100 ml culture to a 250 ml centrifuge bottle, and centrifuge at 12,500 rpm 4°C in the GSA rotor.
4. Decant the supernatant and resuspend the pellet in 20 ml of 1% N-lauryl sarcosine in 1x PBS.
5. Transfer 10 ml of the suspension to each of two Corning CentriStar 50 mL polypropylene (PP) conical tubes (product #4558, maximum RCF is 15,500 x g).
6. Incubate the suspensions at 56°C for two hours.
7. Add 40 ml of absolute ethanol to each tube, and allow to precipitate overnight at -20°C.
8. Centrifuge the ethanol precipitations in a GSA rotor using 50 ml conical adapters (Sorvall part number 03072) at 9,500 rpm (14,678 x g) at 4°C for 30 minutes.
9. Decant the ethanol, and allow the pellets to air dry until there is no ethanol smell.
10. Resuspend each pellet in 3 ml of 100 mM Tris, pH 7.5. The pellets may not completely dissolve, but can be disaggregated fairly well by vortexing. The pellets will likely dissolve during the RNAse digestion step.
11. Add 1 ml of RNAse A solution to each tube and incubate at 37°C for one hour. The volume per tube is now ~4 ml, digesting at 500 µg/ml RNAse A (~2,500 U/ml).
12. Add 4 ml of DNAse solution to each tube, thus doubling the volume and setting the conditions to 100 mM Tris, pH 7.5, 25 mM MgCl2, 5 mM CaCl2, favorable for DNAse digestion. The volume per tube is now ~8 ml, digesting at 500 µg/ml DNAse (~2,000 U/ml).
13. Incubate the tubes at 37°C for one hour.
14. Add 2 ml of proteinase K solution to each tube. The volume is now ~10 ml, digesting at 500 µg/ml proteinase K (>10 U/ml).
15. Incubate the tubes at 56°C for two hours.
16. Transfer the digested samples (~10 ml) to new Corning CentriStar 50 mL polypropylene conical tubes, and add ~40 ml freezer-cold 100% ethanol to each (fill the tube right to the top).
17. Precipitate the samples overnight at -20°C.
18. Collect the precipitants by centrifugation using the GSA rotor with a 50 ml conical adapters at 9,500 rpm (14,678 x g) at 4°C for 30 minutes.
19. Decant the ethanol and re-fill the tubes with freezer-cold 80% ethanol. Agitate the tubes enough to detach the pellet (by briefly vortexing and inverting the tubes several times) and re-centrifuge at 8,000 rpm (10,409 x g) at 4°C for 15 minutes using the GSA rotor and a 50 ml conical adapters.
20. Decant the ethanol:water supernatant, and allow the pellets to air dry until there is no ethanol smell.
21. Resuspend each pellet in 5 ml - 10 ml of a buffer appropriate to intended use.

RNAse solution (5 ml, 2 mg/ml)
10 mg RNAse A (Worthington Biochemical Corp., 4,730 U/mg)

Dissolve in a final volume of 5 ml using 100 mM Tris, pH 7.5.
Store at -20°C.


DNAse solution (17 ml, 1 mg/ml)
17 mg DNAse I (Worthington Biochemical Corp., 3,915 U/mg)

Dissolve in 17 ml of 2× DNAse digestion buffer (100 mM Tris, pH 7.5, 50 mM MgCl2, 10 mM CaCl2).
Store at -20°C


Proteinase K solution (10 ml, 2.5 mg/ml)
25 mg proteinase K (Invitrogen, >20 U/mg)

Dissolve in 5 ml 100 mM Tris, pH 7.5 and 5 ml 2 × DNAse digestion buffer (100 mM Tris, pH 7.5, 50 mM MgCl2, 10 mM CaCl2).
Store at -20°C.

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I understand the protocol uses enzymes to remove RNA and DNA would leave an elevated protein level to address (but thanks for not providing irrelevant links)? With that source and protein from the cells, i'd imagine some level would come down with ethanol precipitation. I suppose one could run a protein assay to see how much remained. I do suppose you could clean it up with some gels.


Can the originator say more about the polysaccharide - molecular weight range, solubility, charge? Is it a specific polymer or a fraction of material you wish to recover.

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True enough, but the final digestion step is >10 U/ml Proteinase K at 56°C for two hours, so much of the protein is destroyed and does not precipitate. When I was working this protocol out, very little remaining host protein was evident on Coomassie- or sliver-stained gels (of course, some PK itself was apparent). An additional digestion with PK and/or Pronase (Calbiochem #53702) did little to alter the appearance of the gels.

I use this procedure rather routinely to isolate various capsular polysaccharides, and have used a variant of it (wherein the original cultures are autoclaved and vortexed rather than treated with 1% sarcosyl), to isolate LPS from Gram-negatives.

To be sure, this does result in a crude PS preparation, sufficient for westerns and the like. On occasions when further purity is needed, I've taken such samples, fractionated them on a gel filtration column (Superpose 6 10/300 GL, GE Healthcare 17-5172-01), dialyzed the fractions against MilliQ water, and lyophilized them.

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It is a high molecular weight PS, about 1500kd, has negative charge and is a specific polymer.

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have you tried some gels?

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i think there not enough literature available for PS gels.
the staining dye , solution etc will all be different from protein gels.

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