I am wondering whether any one have tried to detect EPS on SDS-page. Please give me hints if you have. Thanks a lot.

Mai

Hello Mai:

Here is a good reference where they used SDS-PAGE for detection of exopolysacharides from Burkholderia (formerly Pseudomonas) pseudomallei cultures.


I. STEINMETZ, M. ROHDE, AND B. BRENNEKE. Purification and Characterization of an Exopolysaccharide of Burkholderia (Pseudomonas) pseudomallei. INFECTION AND IMMUNITY, Oct. 1995, p. 39593965 Vol. 63, No. 10.

Abstract:
Burkholderia pseudomallei (basonym Pseudomonas pseudomallei) is the causative organism of melioidosis, a
disease which is recognized as a major public health problem primarily in Southeast Asia and Northern Australia. In this paper, we report on the identification, purification, and characterization of a species-specific exopolysaccharide of B. pseudomallei. After immunization of mice with a B. pseudomallei strain exhibiting
mucoid growth characteristics, we isolated an immunoglobulin G1 monoclonal antibody (MAb) (3015) with specificity for a carbohydrate structure as determined by immunoblotting following sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electron microscopy studies with MAb 3015 revealed reactivity with an exopolysaccharide with a capsule-like appearance in the immunizing strain. All of the mucoid and nonmucoid B. pseudomallei strains tested from geographically different tropical regions were recognized by MAb 3015 in
an enzyme-linked immunosorbent assay or immunoblot, indicating that the exopolysaccharide is constitutively expressed among this species. Intensive testing for cross-reactivity including members of all the Pseudomonas rRNA groups showed no cross-reactivity except in the case of the closely related species Burkholderia mallei. A protocol for purification of the exopolysaccharide which is based principally on mechanical separation from the cell surface followed by repetitive ethanol precipitation steps and finally affinity chromatography using MAb
3015 was established. The exopolysaccharide yielded was of high purity. Gel permeation chromatography was performed, and the molecular mass was estimated to be >150 kDa. Sera from patients with melioidosis were strongly reactive with the purified exopolysaccharide, indicating its in vivo expression and immunogenicity in natural infection. The diagnostic value of the exopolysaccharide and its role in the pathogenesis of disease must still be determined.



Here is the method paraphrased from the full-text article referenced below:

"Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. SDS slab gel electrophoresis was performed as described previously (18). Bacteria grown on agar plates were washed twice with physiological saline and adjusted to a concentration of 5 3 109 cells per ml. After centrifugation in an Eppendorf Biofuge at 10,000 3 g for 5 min, the pellet from 1 ml of the bacterial suspension was suspended in 1 ml of sample buffer (0.0625
MTris-hydrochloride [pH 6.8], 5% 2-mercaptoethanol, 2% SDS, 12.5% glycerol) and heated for 10 min at 1008C. A sample of 20 ml (equivalent to 108 bacteria) was applied to each lane of the gel. Molecular weights were estimated by comparison with a prestained SDS marker kit (MW-SDS-Blue; Sigma). When purified exopolysaccharide or bacterial extracts were examined, 8 mg was applied to each lane of the gel. The blotting procedure was done by previously described methods (18) with the minor modification of using 200 V for 3 h. The nitrocellulose sheet was saturated with 2% (wt/vol) instant dried milk in buffer A overnight. MAb 3015 was purified by affinity chromatography on protein ASepharose (Pharmacia, Uppsala, Sweden) and diluted 1:2,000 in 2% instant dried milk-buffer A. Peroxidase-conjugated goat anti-mouse immunoglobulin
(Jackson Immunoresearch Laboratories) (diluted 1:2,000 in 2% dried milkbuffer A) was used for final development. The substrate was 4-chloro-1-naphthol (Sigma)."


Reference cited in this para-phrased method -

Peters, H., M. Jurs, B. Jann, K. Jann, K. N. Timmis, and D. Bitter-Suermann. 1985. Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide. Infect. Immun. 50:459466.

Here's another reference that may be of some use to you...


G. H. Van Geel-Schutten, E. J. Faber, E. Smit, K. Bonting, M. R. Smith, B. Ten Brink, J. P. Kamerling, J. F. G. Vliegenthart, and L. Dijkhuizen. Biochemical and Structural Characterization of the Glucan and Fructan Exopolysaccharides Synthesized by the Lactobacillus reuteri Wild-Type Strain and by Mutant Strains. Applied and Environmental Microbiology, July 1999, p. 3008-3014, Vol. 65, No. 7

Abstract:
Lactobacillus reuteri LB 121 cells growing on sucrose synthesize large amounts of a glucan (D-glucose) and a fructan (D-fructose) with molecular masses of 3,500 and 150 kDa, respectively. Methylation studies and 13C or 1H nuclear magnetic resonance analysis showed that the glucan has a unique structure consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted alpha -glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was identified as a (2right-arrow6)-beta -D-fructofuranan or levan, the first example of levan synthesis by a Lactobacillus species. Strain LB 121 possesses glucansucrase and levansucrase enzymes that occur in a cell-associated and a cell-free state after growth on sucrose, raffinose, or maltose but remain cell associated during growth on glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sucrose culture supernatants, followed by staining of gels for polysaccharide synthesizing activity with sucrose as a substrate, revealed the presence of a single glucansucrase protein of 146 kDa. Growth of strain LB 121 in chemostat cultures resulted in rapid accumulation of spontaneous exopolysaccharide-negative mutants that had lost both glucansucrase and levansucrase (e.g., strain K-24). Mutants lacking all levansucrase activity specifically emerged following a pH shiftdown (e.g., strain 35-5). Strain 35-5 still possessed glucansucrase and synthesized wild-type glucan.



And here is the paraphrased SDS-PAGE method from this report...

"Gel electrophoresis. SDS-PAGE was performed according to Laemmli (27) by using the Phast System from Pharmacia with 10 to 15% polyacrylamide gels. After activity staining, the gels were silver stained (21). Lysozyme (molecular mass, 14,400), soybean trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (45,000), bovine serum albumin (66,200), and phosphorylase b (97,400) were used as molecular mass references."


References cited by this method...

Heukeshoven, J., and R. Dernick. 1985. Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6:103-112.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680-5.

Thanks a lot for your suggestions, Tony.

I am looking for a simple method that can be used show the eps- phenotype (mutants I am trying to make) and the wild type (eps+) in the bacterial species i am working on.

I could not find any picture in the literature showing the pattern of EPS on polyacrylamide gels.

I do not actually understand the principle behind silver stain method. Thought, it's specific for protein only. It turns out that the method was modified for lipopolysaccharides too. KITTELBERGER R, HILBINK F
SENSITIVE SILVER-STAINING DETECTION OF BACTERIAL LIPOPOLYSACCHARIDES IN POLYACRYLAMIDE GELS
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS 26 (1): 81-86 FEB 1993

People also use PAS (Periodic Acid Schiff) to identify glucose components of EPS. Again, I could not find any image of EPS on SDS-page with PAS staining. Does any one have?

What method do you think is better: PAS or silver?

Thanks a lot in advance.

Mai