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SDS - polyacrylamide gel electrophoresis of lipopolysaccharidesl~* Dit'isiot~of Biologiccrl Sciences, Nrrtiorio/ Reserrrch Corrncil of Crrrlnd(r,Ottan~m,Crrriodrr KIA OR6 Accepted August 18, 1975
RUSSELL, R. R. 8 . . and K. G. JOHNSON.1975. SDS - polyacrylamide gel electrophoresis of 1ipopolysaccharides. Can. J. Mlcrobiol. 21: 2013-2018. Lipopolysaccharides (LPS) prepared from four different species of Neisserirr have been separated by SDS - polyacrylarnide gel electrophoresis. Each LPS possessed a characteristic mobility on gels. Examination of the effect of acrylamide concentration on migration illustrated that the basis of the separation was molecular size, and not intrinsic charge. RUSSELL,R. R. B., et K. G. JOHNSON.1975. SDS - polyacrylamide gel electrophoresis of lipopolysaccharides. Can. J . Microbiol. 21: 2013-2018. Des l~popolysaccharides(LPS) prepares B partlr de quatre especes de Neisserirr ont ete separes par electrophorese sur gel polyacrylamide en presence de SDS. Chaque LPS posskde une mobilite caracteristlque sur les gels. L'examen de la relat~onentre la concentration d'acrylamide et la vitesse de migration B montre que la separation est due a la dimension de la molecule et non pas aux charges electrlques ~ntrinseques.
Lipopolysaccharides (LPS), which comprise a large part of the outer membrane of Gramnegative bacteria, are constructed from repeating units consisting of 'lipid A' to which is attached a 'core' segment of sugar residues. In certain organisms there is an additional polysaccharide '0 chain' (or chains) attached to the core. Although much is now known about the composition of this basic unit in a wide range of organisms (for a review see 5), there is as yet little information available on the way in which the units are assembled into the polymeric form. LPS polymers can be dissociated into subunits by the detergent action of deoxycholate or sodium dodecyl sulphate (SDS). Several workers have investigated the biological properties of such subunits and have estimated their molecular weight by physical techniques based on viscosity measurements and behavior in the ultracentrifuge (1, 7, 8, 10). Olins and Warner (7) have discussed the difficulties inherent in such estimations. Also known is that LPS, dissociated by SDS, migrates in SDS - polyacrylamide gel electrophoresis (9, 11, 14). Although LPS has been reported to behave in a similar manner to proteins in SDS - polyacrylamide gel electrophoresis, its mobility depending on molecular size rather than intrinsic charge, the evidence presented was based on LPS from only a single 'Received July 14, 1975. 2NRCC No. 14973.
organism (14). The resolution of LPS preparations from a number of different nonpathogenic strains of Neisseria and investigation of the basis of their separation is described herein. Materials and Methods Organisms The bacterial species used were Neisseria canis ATCC 14187 (National Research Council of Canada Culture Collection No. 31005), N . caviae ATCC 14659 (NRC 31003), N. pava ATCC 14221 (NRC 3 1010), and a strain of N. sicca (NRC 31018) kindly provided by Dr. J . Kirkland of Proctor and Gamble Ltd. Preparation of LPS The bacteria were grown in a 50-litre stirred fermentor in a defined phosphate buffer - mineral salts - amino acid - vitamin medium developed in this laboratory (6). Cells were freeze-dried and extracted by the hot aqueous phenol procedure of Westphal and Jann (13). LPS was purified by repeated centrifugations at 105 OOOg, and stored frozen in water at a concentration of 5 mg dry weightlml. SDS - Polyacrylarnide Gel Electroplroresis The discontinuous Tris-hydrochloride buffer system of Laemmli (4), which has 0.1% SDS in both the gels and the electrode buffer, was used. Ten-centimetre electrophoresis tubes, 5 mm inside diameter, contained 1.6 ml of separating gel and 0.2 ml of stacking gel. LPS samples contained 25 p1 of the stock (5 mg/ml) solution in 100 p1 of the Laemmli solubilizing buffer. Heating of the sample, either a t 37 "C for 2 h or a t 100°C for 5 min, did not affect the subsequent migration of the LPS. For the protein samples, 10 pg of protein in 100 p1 solubilizing buffer was placed in a boiling water bath for 5 min. The proteins used were lysozyme (Worthington Chemical Corp.), trypsin (Sigma), and pepsin (Sigma). Electro-
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CAN. J. MlCROBllDL. VOL. 21, 1975
by finding the RM of the proteins in gels oi varying acrylamide concentrations, and plottini the logarithm of RM versus gel concentration If charge is the basis of separation, a series 01 parallel lines for a 'charge family' of molecule is obtained. On the other hand, if molecular size Staining Procedures Protein bands were detected by fixing gels for 15 min is the basis of the separation, convergent line in 12.5% trichloroacetic acid (TCA) at 6O0C, staining will be obtained, extrapolating back to meet at ; for 15 rnin at 60 OC in Coomassie blue (2 g Coomassie gel concentration which causes 'minimal sieving brilliant blue in 100 ml glacial acetic acid, 450 ml ethanol, (i.e. a theoretical concentration at which a1 and 450 mI water), then diffusion-destaining in 10% molecules, regardless of size, will migrate un acetic acid. impeded by the gel). The latter case obtains wit1 LPS was detected by fixing in 12.5% TCA and then staining by the periodate-Schiff (PAS) method of Segrest proteins migrating in SDS - polyacrylamide ge and Jackson (12). electrophoresis and, as Fig. 3 shows, in the The relative mobility (RM) of protein or LPS bands is buffer-gel system used in this laboratory, the defined as the distance from the top of the separating gel slopes for a number of different proteins inter to the center of the band, divided by the distance from the top of the separating gel to the point reached by the sect at about 5% acrylamide concentration. bromophenol blue tracking dye. Figure 4 shows the slopes given by the Neisserii LPS preparations. It is readily evident that, likc Cellulose .4ce/a/e Elec/rophoresis Samples (from LPS stock solutions at 5 mglml) were proteins, LPS molecules are separated on the applied to Beckman cellulose acetate strips and subjected basis of their molecular size. Their migration i to electrophoresis using the Beckman R-101 Microzone presumably due to the fact that they bind SDS electrophoresis cell. The buffer used was 0.1 M Tris-HCI (pH 7.5) containing 1% sodium deoxycholate. Electro- as do proteins (7). Although both LPS and proteins give ver! phoresis was carried out at a constant 250 V for 30 min. Cellulose acetate strips were stained in 0.5% Alcian blue similar straight line plots, with convergent line: in 2% acetic acid, and destained in 2% acetic acid. in both cases extrapolating back to meet at z gel concentration in the vicinity of 5%, there arc Results and Discussion obviously differences in the factors determinin! The separation of LPS prepared from four the slopes of the lines. This is illustrated by plot different species of Neisseria on a 15% acrylamide ting data on the mobilities of proteins and LP: gel is shown in Fig. 1. It can be clearly seen that together (Fig. 3) and means that the moleculai each LPS preparation gives a single, somewhat weight of a LPS cannot be estimated from z diffuse band of characteristic mobility. The standard curve prepared from data on thc lower of the two PAS-staining bands in gel 4 is mobilities of proteins of known molecular weigh due to the presence of a small amount of con- For instance, the apparent molecular weight o taminating polysaccharide in the N. sicca LPS N. caviae LPS run on a 10% gel and comparec preparation. with protein standards run under the same con To establish whether the LPS species were ditions is around 18 000, while if the experimen being separated on the basis of their size or their is repeated with 20% gels, a figure close to 800( charge, and to obtain a preliminary indication is obtained. One probable reason for such dis that charge was not the determining factor, crepancies is the fact that disaggregated LP5 preparations were subjected to cellulose acetate consists of long rods (7, lo), as compared witf electrophoresis (Fig. 2). Mobilities of the LPS the more compact conformation of proteins preparations showed no correlation with their The same sort of problem will of course arisc mobilities on SDS - polyacrylamide gels. The with other separation techniques based on 2 surfactant sodium deoxycholate was included in sieving mechanism, such as gel-permeatior the cellulose acetate electrophoresis buffer as, chromatography. in the absence of deoxycholate, most of the LPS The results presented here do not allow an) remained at the point of application. conclusions as to the molecular weight of t h The elegant experiments of Hedrick and Smith LPS isolated from Neisseria, or to compare theil (2) illustrated how the mode of separation of size with values reported by others. The lineal proteins on acrylamide gels may be determined relationship between molecular size and relativc
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Université Laval Bibliotheque on 12/26/14 For personal use only.
phoresis was carried out at 1 mA per tube until the sample had entered the separating gel, then at 3 mA per tube until the bromophenol blue tracking dye was about 5 mm from the bottom of the gel. After removal of the gels from their tubes, the position of the tracking dye was marked by the insertion of a needle dipped in china ink.
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RUSSELL AND JOHNSON: S D S - POLYACRYLAMIDE G E L ELECTROPHORESIS O F LPS
FIG. 1. Electrophoresis of LPS preparations on SDS - polyacrylamide gels containing 15% acr) LPS plrepared from 1, N. canis; 2 , N . cauiae; 3, N . paua; 4, N. sicca.
20 15
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CAN. J. MICROBIOL. VOL. 21, 1975
FIG.2. Electrophoresis of LPS preparations on cellulose acetate. 1 , N. canis; 2, N . caviae; 3, N. fla~ia; 4 , N. sicca. The anode was toward the bottom of the tubes.
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RUSSELL AND JOHNSON: SDS - POLYACRYLAMIDE G E L ELECTROPHORESIS O F LPS
20 17
GEL CONCENTRATION % GEL CONCENTRATION %
FIG. 3. The effect of acrylamide concentration on the mobility of proteins in SDS - polyacrylamide gel electrophoresis. Pepsin ( a ) , trypsin (O), and lysozyme (A). The mobility of LPS from N . caoiae is shown by the dotted line (data from Fig. 4).
mobility, however, does indicate that SDS polyacrylamide gel electrophoresis will be useful in determining the sizes of LPS subunits once standard LPS preparations, for which the molecular weight has been determined by other methods, become available. Electrophoresis of LPS preparations may be of some value in taxonomic studies. Examination of LPS from nine different species of Neisseria has revealed that LPS with the same core-oligosaccharide structures possessed identical electrophoretic mobilities (3). SDS - polyacrylamide gel electrophoresis has been of considerable value in the investigation of N . cinereae, an organism which possesses two distinct LPS types (3, unpublished).
Acknowledgment We are grateful to Dr. I. J. McDonald for his assistance in bacterial cultivation.
FIG. 4. The effect of acrylamide concentration on the mobility of LPS prepared from N. canis (A), N . caviae (O), N. flaoa ( a ) , and N . sicca (0).
1. BEER, H., T . STACHELIN, H. DOUGLAS,and A. I. BRAUDE. 1965. Relationship between particle size and biological activity ofE. coli Boivin endotoxin. J. Clin. Invest. 44: 592-602. 2. HEDRICK, J. L., and A. J. SMITH.1968. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126: 155-164. 3. JOHNSON, K. G., M. B. PERRY,I. J . M C D O N A L D , ~ ~ ~ R. R. B. RUSSELL.1975. Cellular and free lipopolysaccharides of some species of Neisseria. Can. J . Microbiol. This issue. 4. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. 5. L~JDERITZ, O., 0 . WESTPHAL, A. M. STAUB,and H. N I K A I D O 1971. . Isolation and chemical and immunological characterization of bacterial lipopolysaccharides. In Microbial toxins. Vol. IV. Edited by G. Weinbaum, S. Kadis, and S. J. Ajl. Academic Press, Inc., New York. pp. 145-233. 6. MCDONALD, I. J., and K. G. JOHNSON. 1975. Nutritional requirements of some non-pathogenic Neisseria growth in simple synthetic media. Can. J . Microbial. 21: 1198-1204. S , L., and R. C . WARNER. 1967. Physiochemi7. O L ~ N A. cal studies on a lipopolysaccharide from the cell wall
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CAN. .I.MICROBIOL. VOL. 21, 1975
of Azotohtrcter ~ ~ i t ~ l a t l dJi.i . Biol. Chem. 242: 4994-5001. 12. OROSZLAN, S. I.. and P. T. MORA.1963. Dissociation and reconstitution of an endotoxin. Biochem. Biophys. Res. Commun. 12: 345-349. , PARISI,and J . OSBORN.M. J . , J . E. G A N D E RE. CARSON.1972. Mechanism of assembly of the outer t y p h i t ~ ~ r r r i ~J.r nBiol. l . Chem. membrane ofStrlti~o~~ellrr 13. 247: 3962-3972. R I B I ,E., R. L. A N A C K E R R., BROWN.W. T. HASKINS, B. MALMGREN, K . C . M I L N E Rand , J. A. RUDBACH. 1966. Reaction of endotoxin and surfactants. I. Physical and biological properties of endotoxin treated with sodium deoxycholate. J. Bacterial. 92: 1493-1509. ROTHFIELD, L., and M. PEARLMAN-KOTHENCZ.14. 1969. Synthesis and assembly of bacterial membrane components. A lipopolysaccharide-phospholipid pro-
tein complex excreted by living bacteria. J . Mol. Biol 44: 477-492. 1972. Molecula SEGREST,J . P.. and R. L. JACKSON. weight determination of glycoproteins by polyac rylamide gel electrophoresis in sodium dodecyl sul phate. 111 Methods in enzymology. Vol. XXVIIl Edited by N . Ginsburg. Academic Press, Inc., Neb York. pp. 54-63. WESTPHAL, O., and K. J A N N . 1965. Bacteria lipopolysaccharides. Extraction with phenol-wate and further applications o f the procedure. I11 Method l R. L in carbohydrate chemistry. Vol. V. E r l i t ~ r by Whistler and M. L. Wolfrom. Academic Press, Inc. New York. pp. 83-91. ZOLLINGER, W. D., D. L. KASPER,B. J. VELTRI,an, M. S. ARTENSTEIN. 1972. Isolation and characteriza tion of a native cell wall complex from Neisserir trletlitlgitidis. Infect. Immun. 6 : 835-851.
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This article has been cited by: 1. Kerstin Ludwig, Enke Grabhorn, Martin Bitzan, Christoph Bobrowski, Markus J Kemper, Ingo Sobottka, Rainer Laufs, Helge Karch, Dirk E Müller-Wiefel. 2002. Saliva IgM and IgA Are a Sensitive Indicator of the Humoral Immune Response to Escherichia coli O157 Lipopolysaccharide in Children with Enteropathic Hemolytic Uremic Syndrome. Pediatric Research 52, 307-313. [CrossRef] 2. Roy A Dalmo, Arild A Kjerstad, Siri M Arnesen, Peter S Tobias, Jarl Bøgwald. 2000. Bath exposure of Atlantic halibut (Hippoglossus hippoglossus L.) yolk sac larvae to bacterial lipopolysaccharide (LPS): Absorption and distribution of the LPS and effect on fish survival. Fish & Shellfish Immunology 10, 107-128. [CrossRef] 3. Stephen G. Wilkinson. 1996. Bacterial lipopolysaccharides—Themes and variations. Progress in Lipid Research 35, 283-343. [CrossRef] 4. Brigitte Brake, Clara Larcher, Thomas F. Schulz, Wolfgang Prodinger, Manfred P. Dierich. 1992. Species Specific Monoclonal Antibodies to Bacteroides fragilis Lipopolysaccharide Protect Mice from Severe Infection. Zentralblatt für Bakteriologie 277, 320-328. [CrossRef] 5. Robin Sandlin, Daniel C. Stein. 1991. Structural heterogeneity of the lipopolysaccharides of the Neisseriaceae. FEMS Microbiology Letters 90:10.1111/fml.1991.90.issue-1, 69-72. [CrossRef] 6. Alan J. Lesse, Anthony A. Campagnari, William E. Bittner, Michael A. Apicella. 1990. Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Journal of Immunological Methods 126, 109-117. [CrossRef] 7. D. J. Hampson, J. R. L. Mhoma, B. Combs. 1989. Analysis of lipopolysaccharide antigens of Treponema hyodysenteriae. Epidemiology and Infection 103, 275. [CrossRef] 8. P. Ibsen, S. Møller, I. Heron. 1988. Lipopolysaccharides in a traditional pertussis vaccine. Journal of Biological Standardization 16, 299-309. [CrossRef] 9. János Samu, Enikö Kováts, Vinh Nguyen, Tibor Keler, Alois Nowotny, Richard T. Coughlin. 1988. Thin-layer chromatography of endotoxins, their derivatives and contaminants. Journal of Chromatography A 435, 167-183. [CrossRef] 10. Blair A. Sowa, Richard P. Crawforda, Fred C. Heck, John D. Williams, Albert M. Wu, Katherine A. Kelly, L. Garry Adams. 1986. Size, charge and structural heterogeneity ofBrucella abortus lipopolysaccharides demonstrated by two-dimensional gel electrophoresis. Electrophoresis 7:10.1002/elps.v7:6, 283-288. [CrossRef] 11. MATTI K. VILJANEN, LINNÉA LINKO, PERTTI ARSTILA, OLLI-PEKKA LEHTONEN, ANDREJ WEINTRAUBMonoclonal Antibodies to the Lipopolysaccharide and Capsular Polysaccharide of Bacteroides fragilis 119-142. [CrossRef] 12. Michio Ohta, Joseph Rothmann, Enikö Kovats, Phuc Hong Pham, Alois Nowotny. 1985. Biological Activities of Lipopolysaccharide Fractionated by Preparative Acrylamide Gel Electrophoresis. Microbiology and Immunology 29:10.1111/ mim.1985.29.issue-1, 1-12. [CrossRef] 13. EYVIND RDAHL, JOHAN A. MAELAND. 1984. AFFINITY CHROMATOGRAPHY FOR PURIFICATION OF ANTIBODIES TO NEISSERIA GONORRHOEAE AND NEISSERIA MENINGITIDIS LIPOPOLYSACCHARIDES. Acta Pathologica Microbiologica Scandinavica Series C: Immunology 92C, 247-254. [CrossRef] 14. EYVIND RØDAHL, JOHAN A. MAELAND. 1983. ANTIBODY RESPONSE IN RABBITS TO GONOCOCCAL LIPOPOLYSACCHARIDE-BOVINE SERUM ALBUMIN CONJUGATES. Acta Pathologica Microbiologica Scandinavica Series B: Microbiology 91B, 285-289. [CrossRef] 15. D.L. Diedrich, A.R. Domenico, J.A. Fralick. 1983. Influence of urea on the resolution of lipopolysaccharides in sodium dodecylsulfate polycrylamide gels. Journal of Microbiological Methods 1, 245-251. [CrossRef] 16. Penny J. HITCHCOCK. 1983. Aberrant Migration of Lipopolysaccharide in Sodium Dodecyl Sulfate/Poyacrylamide Gel Electrophoresis. European Journal of Biochemistry 133:10.1111/ejb.1983.133.issue-3, 685-688. [CrossRef] 17. P COLEMANChapter 12 Lipopolysaccharides 281-285. [CrossRef] 18. Chao-Ming Tsai, Carl E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Analytical Biochemistry 119, 115-119. [CrossRef] 19. Sissel F. Rø, Guri Eggset, Ole-Jan Iversen, Johan A. Maeland. 1980. CHARACTERISTICS OF ANTISERA AGAINST PERIODATE-RESISTANT MEMBRANE ANTIGENS FROM. NEISSERIA GONORRHOEAE. Acta Pathologica Microbiologica Scandinavica Section B Microbiology 88B, 329-334. [CrossRef]
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20. Robert C. GOLDMAN, Loretta LEIVE. 1980. Heterogeneity of Antigenic-Side-Chain Length in Lipopolysaccharide from Escherichia coli 0111 and Salmonella typhimurium LT2. European Journal of Biochemistry 107:10.1111/ejb.1980.107.issue-1, 145-153. [CrossRef] 21. J. N. Saddler, R. Parton, A. C. Wardlaw. 1979. Degradation of bacterial lipopolysaccharide by gut juice of the snailHelix pomatia. Experientia 35, 494-495. [CrossRef]
SDS - polyacrylamide gel electrophoresis of lipopolysaccharidesl~* Dit'isiot~of Biologiccrl Sciences, Nrrtiorio/ Reserrrch Corrncil of Crrrlnd(r,Ottan~m,Crrriodrr KIA OR6 Accepted August 18, 1975
RUSSELL, R. R. 8 . . and K. G. JOHNSON.1975. SDS - polyacrylamide gel electrophoresis of 1ipopolysaccharides. Can. J. Mlcrobiol. 21: 2013-2018. Lipopolysaccharides (LPS) prepared from four different species of Neisserirr have been separated by SDS - polyacrylarnide gel electrophoresis. Each LPS possessed a characteristic mobility on gels. Examination of the effect of acrylamide concentration on migration illustrated that the basis of the separation was molecular size, and not intrinsic charge. RUSSELL,R. R. B., et K. G. JOHNSON.1975. SDS - polyacrylamide gel electrophoresis of lipopolysaccharides. Can. J . Microbiol. 21: 2013-2018. Des l~popolysaccharides(LPS) prepares B partlr de quatre especes de Neisserirr ont ete separes par electrophorese sur gel polyacrylamide en presence de SDS. Chaque LPS posskde une mobilite caracteristlque sur les gels. L'examen de la relat~onentre la concentration d'acrylamide et la vitesse de migration B montre que la separation est due a la dimension de la molecule et non pas aux charges electrlques ~ntrinseques.
Lipopolysaccharides (LPS), which comprise a large part of the outer membrane of Gramnegative bacteria, are constructed from repeating units consisting of 'lipid A' to which is attached a 'core' segment of sugar residues. In certain organisms there is an additional polysaccharide '0 chain' (or chains) attached to the core. Although much is now known about the composition of this basic unit in a wide range of organisms (for a review see 5), there is as yet little information available on the way in which the units are assembled into the polymeric form. LPS polymers can be dissociated into subunits by the detergent action of deoxycholate or sodium dodecyl sulphate (SDS). Several workers have investigated the biological properties of such subunits and have estimated their molecular weight by physical techniques based on viscosity measurements and behavior in the ultracentrifuge (1, 7, 8, 10). Olins and Warner (7) have discussed the difficulties inherent in such estimations. Also known is that LPS, dissociated by SDS, migrates in SDS - polyacrylamide gel electrophoresis (9, 11, 14). Although LPS has been reported to behave in a similar manner to proteins in SDS - polyacrylamide gel electrophoresis, its mobility depending on molecular size rather than intrinsic charge, the evidence presented was based on LPS from only a single 'Received July 14, 1975. 2NRCC No. 14973.
organism (14). The resolution of LPS preparations from a number of different nonpathogenic strains of Neisseria and investigation of the basis of their separation is described herein. Materials and Methods Organisms The bacterial species used were Neisseria canis ATCC 14187 (National Research Council of Canada Culture Collection No. 31005), N . caviae ATCC 14659 (NRC 31003), N. pava ATCC 14221 (NRC 3 1010), and a strain of N. sicca (NRC 31018) kindly provided by Dr. J . Kirkland of Proctor and Gamble Ltd. Preparation of LPS The bacteria were grown in a 50-litre stirred fermentor in a defined phosphate buffer - mineral salts - amino acid - vitamin medium developed in this laboratory (6). Cells were freeze-dried and extracted by the hot aqueous phenol procedure of Westphal and Jann (13). LPS was purified by repeated centrifugations at 105 OOOg, and stored frozen in water at a concentration of 5 mg dry weightlml. SDS - Polyacrylarnide Gel Electroplroresis The discontinuous Tris-hydrochloride buffer system of Laemmli (4), which has 0.1% SDS in both the gels and the electrode buffer, was used. Ten-centimetre electrophoresis tubes, 5 mm inside diameter, contained 1.6 ml of separating gel and 0.2 ml of stacking gel. LPS samples contained 25 p1 of the stock (5 mg/ml) solution in 100 p1 of the Laemmli solubilizing buffer. Heating of the sample, either a t 37 "C for 2 h or a t 100°C for 5 min, did not affect the subsequent migration of the LPS. For the protein samples, 10 pg of protein in 100 p1 solubilizing buffer was placed in a boiling water bath for 5 min. The proteins used were lysozyme (Worthington Chemical Corp.), trypsin (Sigma), and pepsin (Sigma). Electro-
2014
CAN. J. MlCROBllDL. VOL. 21, 1975
by finding the RM of the proteins in gels oi varying acrylamide concentrations, and plottini the logarithm of RM versus gel concentration If charge is the basis of separation, a series 01 parallel lines for a 'charge family' of molecule is obtained. On the other hand, if molecular size Staining Procedures Protein bands were detected by fixing gels for 15 min is the basis of the separation, convergent line in 12.5% trichloroacetic acid (TCA) at 6O0C, staining will be obtained, extrapolating back to meet at ; for 15 rnin at 60 OC in Coomassie blue (2 g Coomassie gel concentration which causes 'minimal sieving brilliant blue in 100 ml glacial acetic acid, 450 ml ethanol, (i.e. a theoretical concentration at which a1 and 450 mI water), then diffusion-destaining in 10% molecules, regardless of size, will migrate un acetic acid. impeded by the gel). The latter case obtains wit1 LPS was detected by fixing in 12.5% TCA and then staining by the periodate-Schiff (PAS) method of Segrest proteins migrating in SDS - polyacrylamide ge and Jackson (12). electrophoresis and, as Fig. 3 shows, in the The relative mobility (RM) of protein or LPS bands is buffer-gel system used in this laboratory, the defined as the distance from the top of the separating gel slopes for a number of different proteins inter to the center of the band, divided by the distance from the top of the separating gel to the point reached by the sect at about 5% acrylamide concentration. bromophenol blue tracking dye. Figure 4 shows the slopes given by the Neisserii LPS preparations. It is readily evident that, likc Cellulose .4ce/a/e Elec/rophoresis Samples (from LPS stock solutions at 5 mglml) were proteins, LPS molecules are separated on the applied to Beckman cellulose acetate strips and subjected basis of their molecular size. Their migration i to electrophoresis using the Beckman R-101 Microzone presumably due to the fact that they bind SDS electrophoresis cell. The buffer used was 0.1 M Tris-HCI (pH 7.5) containing 1% sodium deoxycholate. Electro- as do proteins (7). Although both LPS and proteins give ver! phoresis was carried out at a constant 250 V for 30 min. Cellulose acetate strips were stained in 0.5% Alcian blue similar straight line plots, with convergent line: in 2% acetic acid, and destained in 2% acetic acid. in both cases extrapolating back to meet at z gel concentration in the vicinity of 5%, there arc Results and Discussion obviously differences in the factors determinin! The separation of LPS prepared from four the slopes of the lines. This is illustrated by plot different species of Neisseria on a 15% acrylamide ting data on the mobilities of proteins and LP: gel is shown in Fig. 1. It can be clearly seen that together (Fig. 3) and means that the moleculai each LPS preparation gives a single, somewhat weight of a LPS cannot be estimated from z diffuse band of characteristic mobility. The standard curve prepared from data on thc lower of the two PAS-staining bands in gel 4 is mobilities of proteins of known molecular weigh due to the presence of a small amount of con- For instance, the apparent molecular weight o taminating polysaccharide in the N. sicca LPS N. caviae LPS run on a 10% gel and comparec preparation. with protein standards run under the same con To establish whether the LPS species were ditions is around 18 000, while if the experimen being separated on the basis of their size or their is repeated with 20% gels, a figure close to 800( charge, and to obtain a preliminary indication is obtained. One probable reason for such dis that charge was not the determining factor, crepancies is the fact that disaggregated LP5 preparations were subjected to cellulose acetate consists of long rods (7, lo), as compared witf electrophoresis (Fig. 2). Mobilities of the LPS the more compact conformation of proteins preparations showed no correlation with their The same sort of problem will of course arisc mobilities on SDS - polyacrylamide gels. The with other separation techniques based on 2 surfactant sodium deoxycholate was included in sieving mechanism, such as gel-permeatior the cellulose acetate electrophoresis buffer as, chromatography. in the absence of deoxycholate, most of the LPS The results presented here do not allow an) remained at the point of application. conclusions as to the molecular weight of t h The elegant experiments of Hedrick and Smith LPS isolated from Neisseria, or to compare theil (2) illustrated how the mode of separation of size with values reported by others. The lineal proteins on acrylamide gels may be determined relationship between molecular size and relativc
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Université Laval Bibliotheque on 12/26/14 For personal use only.
phoresis was carried out at 1 mA per tube until the sample had entered the separating gel, then at 3 mA per tube until the bromophenol blue tracking dye was about 5 mm from the bottom of the gel. After removal of the gels from their tubes, the position of the tracking dye was marked by the insertion of a needle dipped in china ink.
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RUSSELL AND JOHNSON: S D S - POLYACRYLAMIDE G E L ELECTROPHORESIS O F LPS
FIG. 1. Electrophoresis of LPS preparations on SDS - polyacrylamide gels containing 15% acr) LPS plrepared from 1, N. canis; 2 , N . cauiae; 3, N . paua; 4, N. sicca.
20 15
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CAN. J. MICROBIOL. VOL. 21, 1975
FIG.2. Electrophoresis of LPS preparations on cellulose acetate. 1 , N. canis; 2, N . caviae; 3, N. fla~ia; 4 , N. sicca. The anode was toward the bottom of the tubes.
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RUSSELL AND JOHNSON: SDS - POLYACRYLAMIDE G E L ELECTROPHORESIS O F LPS
20 17
GEL CONCENTRATION % GEL CONCENTRATION %
FIG. 3. The effect of acrylamide concentration on the mobility of proteins in SDS - polyacrylamide gel electrophoresis. Pepsin ( a ) , trypsin (O), and lysozyme (A). The mobility of LPS from N . caoiae is shown by the dotted line (data from Fig. 4).
mobility, however, does indicate that SDS polyacrylamide gel electrophoresis will be useful in determining the sizes of LPS subunits once standard LPS preparations, for which the molecular weight has been determined by other methods, become available. Electrophoresis of LPS preparations may be of some value in taxonomic studies. Examination of LPS from nine different species of Neisseria has revealed that LPS with the same core-oligosaccharide structures possessed identical electrophoretic mobilities (3). SDS - polyacrylamide gel electrophoresis has been of considerable value in the investigation of N . cinereae, an organism which possesses two distinct LPS types (3, unpublished).
Acknowledgment We are grateful to Dr. I. J. McDonald for his assistance in bacterial cultivation.
FIG. 4. The effect of acrylamide concentration on the mobility of LPS prepared from N. canis (A), N . caviae (O), N. flaoa ( a ) , and N . sicca (0).
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