I have run a 12% SDS-PAGE/8M Urea gel for my bacterial EPS sample and have got bands for the polysaccharide after silver staining it. I need to elute the desired band for further analysis of the sugars. How can the band be eluted manually so that the sugars do not get degraded?
The methodology employed to separate polysaccharides in EPS seems to be improper. It is better to separate the different polysaccharides through gel filtration and then analyse different fractions.
You can cut with the blade the bands equivalent to the staine one; the gel get pulverized/crushed; the fined solid material mixed with Biogel P-2 gel (about 1:1 volume) which allows separation of liquid from the gel mztrix by centrifugation . You should wash the gel mix with the buffer or water. Then the eluted polysaccharide could be dialized againt water or any buffer you are going to use. Of course the dialyzing membrain should be met the required mw cut-off for the polysaccharide. The multiple bands should be pooled together to increaes the amount depending upon the sensitivity of the next analyzing instrument. Normally the amount of the polysaccharide in a gel as detected by stainf method you used is very tiny amount; very dificult to work for further analyses unless multiple samples combined.
Unfortunately, it is not possible to elute EPS from gels. In addition, after silver staining your polysaccharide has been oxidised (i.e. your sugar has already been degraded) and the resulting aldehyde groups conjugated to the silver stain so the residues are not longer available for analysis.
For analysis, you will have to purify your EPS using chromatography (usually a combination of anion exchange and size exclusion, depending on your individual EPS).
Assuming the polysaccharide is charged. It can be 'eluted' from the gel by placing gel slice in snake skin dialysis tubing (check the mw cut off first) filled with TBE buffer. Place in an electrophoresis tank also filled with TBE similar like you would for an agarose gel and run a current through it. The polysaccharide should move out of the gel slice and into the buffer contained in the dialysis tubing. The polysaccharide can then be precipitated from the buffer using 3 volumes of absolute ethanol, washed with 70% ethanol and dried. Once it is dry resuspend in double distilled water pH8.0.
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