Extracellular Polysaccharides, Lipopolysaccharides and Glycolipids

Purification:
Many purification methods for extracellular and membrane bound saccharides are well worked out.  If you are having trouble isolating or purifying your compound, we are happy to help.  Methods of purification we can currently perform include:

Size Exclusion Chromatography – Our lab currently has both Superose 12 and Superdex 75 columns to purify saccharides from 1000 to several hundred thousand daltons molecular weight.  For smaller compounds we have a BioGel P2 column capable of resolving oligosaccharides.  Data from this type of analysis can often be used for size determination and proof of purity.

Ion Exchange Chromatography – Many extracellular and membrane bound saccharides carry significant negative charge.  This allows for efficient purification using a DEAE solid phase with either stepwise or gradient elution for purification.

Reverse Phase Chromatography - If your molecule possesses a hydrophobic aglycone, we have an Agilent reverse phase HPLC system that can be used for purification of up to 1mg of sample.

Fractionation of crude extract on Agilent Eclipse reverse phase column using acetonitrile gradient in water.

Lipopolysaccharides Isolation and Purification – We offer a variety of LPS isolation techniques including (but by no means limited to) phenol/water, PCP, detergent extraction, phenol TEA EDTA and enzymatic methods.  If you are not sure which of these techniques is right for you, we can evaluate them head to head using only 10mg of cells per test to ensure that the initial extract is optimized for both purity and yield.  Note that we can also test for minor components left behind in cell pellets or phenol phases at no extra charge.  For optimum results with composition and linkage analysis, we recommend a minimum of 120mg dry cell mass starting material.

Comparison of various extraction methods for LPS extraction from  Cyanobacteria.  Gel on left is silver stained while the gel on the right uses the Molecular Probes ProQ Emerald Stain.  Lane 1) E. coli O55:B5 standard Lane 2) Phenol water aqueous ultraprecipitate Lane 3) Phenol water aqueous retentate from ultrafiltration Lane 4) Detergent extract magnesium precipitate Lane 5) Pellet from phenol water extraction showing that some material is left behind it is similar in nature to the extracted material Lane 6) Phenol phase from phenol water extraction showing low molecular weight components left behind in the extraction Lane 7) E. coli complete core (Ra) LPS

Proof of Purity:
Often overlooked, assurance that a sample contains only a single saccharide is crucial to interpreting data from other analyses.  To this end, we offer two kinds of service:

SDOC PAGE – Most useful for LPS and CPS this method offers high-resolution size separation of molecules up to approximately 100kD molecular weight.  Methods of staining include silver staining, the nondestructive zinc imidazole stain and the Molecular Probes ProQ Emerald stain.  We also have a special silver staining method which can help differentiate between LPS and CPS.

Size Exclusion Chromatography – Also useful in purification, a single, symmetrical peak in size exclusion chromatography is prima facie evidence of purity.  See the purification section for more information.