Lipopolysaccharides
from Escherichia coli 055:B5 Product Number L 2880 Storage Temperature 2-8 °C
Product Description
Synonym: LPS
This product is phenol extracted from E. coli rotype O55:B5. The source strain is CDC 1644-70.
The LPS O55:B5 has been ud to stimulate human peritoneal macrophages at 1 ng/ml22 and to stimulate equine peritoneal macrophages at 1-100 ng/ml23. Lipopolysaccharides (LPS) are characteristic components of the cell wall of Gram negative bacteria; they are not found in Gram positive bacteria. They are localized in the outer layer of the membrane and are, in noncapsulated strains, expod on the cell surface. They contribute to the integrity of the outer membrane, and protect the cell against the action of bile salts and lipophilic antibiotics.1
Lipopolysaccharides are made up of a hydrophobic lipid (lipid A, which is responsible for the toxic properties of the molecule), a hydrophilic core polysaccharide chain, and a hydrophilic O-antigenic polysaccharide side chain. In most cas, O-specific chains are built of repeating units of oligosacchar
ides which exhibit a strain-specific structural diversity. The sugar constituents, their quence, and their mode of linkage determine the rological O specificity of respective strains. They are the main determinants of the classifications of the rotypes of Salmonella species. The diversity of O chains in Enterobacteriaceae may have developed during evolution to allow enteric bacterial to escape the host’s immune system by developing new specificities on their cell surface (specific to the bacterial rotype).1Since lipopolysaccharides confer antigenic properties on the cell, they have been termed O antigens. As the main antigen, lipopolysaccharides are involved in various host-parasite interactions. They em to protect Gram negative bacteria from phagocytosis and lysis.1 Bacteria with common rotypes have surface antigens (group O, group H, or LPS) which generate the same antibody respon. Examples of rotypes are O55:B5 and O26:B6 for the E. coli bacterium. The designations are immunological classifications, which specify which antibody recognized which strains. Different strains may have some common antigenic determinants.
If a wild strain of bacterium is irradiated with UV light
or expod to mutagenic compounds, it will mutate. The few mutations that are not lethal result in viable mutants (rough strains) which are generally not found in nature, and which posss some unique characteristics. The genes that encode lipopolysaccharide formation may also be altered in t
he mutants, and LPS with shorter polysaccharide chains may be formed. Ra, Rb, Rc, Rd, Re, etc. (where a, b, c, designate 1st, 2nd, 3rd, degree, respectively) designate the polysaccharide length of a given LPS. Ra and Re designate the mutants with the longest and shortest chain lengths, respectively.2 The most extreme mutants are the Re mutants which produce an LPS which is made up of Lipid A and 3-deoxy-D-manno-octulosonic acid
(2-Keto-3-deoxyoctonate, KDO) as the sole constituent of the core.2 Lipid A and lipopolysaccharides from rough strains are tested for KDO content.3
Purified endotoxin is generally referred to as lipopolysaccharide or LPS, to distinguish it from the more natural complexed cell membrane associated form. The core portion of the polysaccharide chain is common to LPS from wild and mutant bacterial strains.
Removal by hydrolysis of the polysaccharide chain from LPS produces lipid A, either as the naturally occurring, cytotoxic diphosphoryl form4 or the less toxic, monophosphoryl form.5,6 The longer the p
olysaccharide chain is, the longer and more difficult the hydrolysis. LPS with a long polysaccharide chain has a relatively low lipid A content, which must be purified from a large amount of hydrolysis byproducts (oligosaccharides and saccharide monomers). Thus, the yield of lipid A is low and recovery is poor. LPS with a short polysaccharide chain (LPS from mutant bacteria) is therefore ud to produce lipid A products. Removal of the fatty acid portions of lipid A results in a detoxified LPS7 with an endotoxin level about
10,000 times lower than that of the parent LPS.
The molecular structure of LPS has been studied.8,9 Since LPS is heterogeneous and tends to form aggregates of varying sizes, the molecular weight is not very meaningful. However, there is a reported range of 1-4 million or greater. When the LPS is treated with SDS and heat, the molecular weight is in the range of 50 to 100 kDa. In their purest form, in the prence of strong surface active agents, and in the abnce of divalent cations, bacterial endotoxins consist of 10-20 kDa macromolecules. In the abnce of surface active agents and in the prence of divalent cation questering agents such as EDTA, LPS is believed to arrange itlf into a micellar structure with a molecular weight of approximately
1,000 kDa. This is the smallest form of bacterial LPS that is likely to exist in aqueous liquids. In the prence of divalent cations such as Ca2+ and Mg2+, a bilayer structure appears to exist that pass through
a 0.2 µm membrane, but does not pass through a
0.025 µm membrane. LPS vesicles up to 0.1 µm in diameter may also be formed in water in the prence of divalent cations. The lf aggregation of LPS is generally a function of the lipid A component of the molecule, which also confers the ability to bind to hydrophobic surfaces.
LPS can be prepared by TCA,10 phenol,11 or phenol-chloroform-petroleum ether (for rough strains)12 extraction. The TCA extracted lipopolysaccharides are structurally similar to the phenol extracted ones. Their electrophoretic pattern and endotoxicity are similar. The main differences are in the amounts of nucleic acid and protein contaminations. The TCA extract contains approximately 2% RNA and approximately 10% denatured proteins. The phenol extract contains up to 60% RNA and less than 1% protein. Purification by gel filtration chromatography removes much of protein prent in the phenol-extracted LPS, but leaves a product that still contains 10-20% nucleic acids. Further purification using ion exchange chromatography, will yield an LPS product which contains <1% protein and <1% RNA. Sigma offers LPS with various levels of protein and/or RNA.
Sigma's lipopolysaccharides contain endotoxin levels of not less than 500,000 EU (endotoxin units)/mg unless otherwi noted. One nanogram of endotoxin is equivalent to 5 EU (Limulus lysate assay) and 10 EU (chromogenic assay).
LPS preparations are ud extensively for rearch in the elucidation of LPS structure,13 metabolism,14 immunology,15 physiology,16 toxicity,17 and biosynthesis.18 They have also been ud to induce synthesis and cretion of growth promoting factors such as interleukins.19
FITC (fluorescein isothiocyanate), TRITC (tetramethyrhodamine isothiocyanate), and TNP (trinitrophenyl) conjugates have been prepared by reacting LPS with either FITC, TRITC or
2,4,6-trinitrobenzenesulfonic acid, respectively.20
They are ud in rearch on the T-independent B cell immune respon to bacterial LPS.20
Precautions and Disclaimer
For Laboratory U Only. Not for drug, houhold or other us.
Preparation Instructions
The product is soluble in water (5 mg/ml) or cell culture medium (1 mg/ml) yielding a hazy, faint yellow solution. A more concentrated, though still hazy, solution (20 mg/ml) has been achieved in aqueous saline after vortexing and warming to 70-80 °C.21 Lipopolysaccharides are molecules that form micelles in every solvent. Hazy solutions are obrved in water and phosphate buffered saline. Organic solvents do not give clearer solutions. Methanol yields a turbid suspension with floaters, while water yields a homogeneously hazy solution.
For cell culture u, LPS should be reconstituted by adding 1 ml of sterile balanced salt solution or cell culture medium to a vial (1 mg) and swirling gently until the powder dissolves. Solutions can be further diluted to the desired working concentration with additional sterile balanced salt solutions or cell culture media.
Storage/Stability
Solutions at 1 mg/ml in buffer or culture medium are stable for approximately one month at 2-8 °C. Frozen aliquots can be stored up to 2 years. Repeated
freeze/thaw cycles are not recommended. Solutions should be stored in silanized containers, since LPS can bind to plastics and certain types of glass (especially at concentrations of <0.1 mg/ml). If th
e LPS concentration is >1 mg/ml, adsorption to the sides of the vial is negligible. If glass containers are ud, solutions should be vortexed for at least 30 minutes to redissolve the adsorbed product.
References
1. Mayer, H. et al., Analysis of Lipopolysaccharides
of Gram-Negative Bacteria. Methods in
Microbiology 18, 157-207 (1985).
2. Raetz, C. R. H., Biochemistry of Endotoxins.
礼物袋怎么折Annu. Rev. Biochem. 59, 129-170 (1990).
3. Cynkin, M. A., Estimation of 3-Deoxy sugars by
means of the Manonaldehyde-Thibarbituric Acid
Reaction. Nature, 186, 155 (1960).
4. Qureshi, N., et al., Position of ester groups in the
lipid A backbone of lipopolysaccharides obtained
from Salmonella typhimurium. J. Biol. Chem.,
258(21), 12947-12951 (1983).
5. Chang C. M., and Nowotny, A., Relation of
四远香Structure to Function in Bacterial O-antigens VII.
Endotoxicity of “lipid A.” Immunochem., 12, 19
(1975).
6. Qureshi N., and Takayama, N., Purification and
structural determination of nontoxic lipid A
obtained from the lipopolysaccharide of
Salmonella typhimurium.J. BioI. Chem., 257,
心情突然很低落
11808-11815 (1982).
7. Ding H. F., et al., Protective immunity induced in
mice by detoxified Salmonella lipopolysaccharide.
J. Med. Microbiol., 31(2), 95-102 (1990).
8. Jann, B., et al., Heterogeneity of
lipopolysaccharides. Analysis of polysaccharide
chain lengths by sodium dodecylsulfate-
polyacrylamide gel electrophoresis. Eur. J.
Biochem., 60, 239-246 (1975).
9. Leive, L., and Morrison, D. C., Isolation of
Lipopolysaccharides from Bacteria. Methods in
Enzymology, 28, 254-262 (1972).
10. Staub, A. M., Bacterial Lipido-protino-
polysaccharides (‘O’ Somatic Antigens) Extraction with Trichloroacetic Acid. Methods in
Carbohydrate Chem., 5, 92-93 (1965). 11. Westphal, O., and Jann, K., Bacterial
Lipopolysaccharides Extraction with Phenol-Water and Further Applications of the Procedure.
Methods in Carbohydrate Chem., 5, 83-91 (1965).
文明寝室12. Galanos, C., et al., A new method for the
extraction of R lipopolysaccharides. Eur. J.
Biochem., 9(2), 245-249 (1969).
13. Strain, S. M., et al., Characterization of
lipopolysaccharide from a heptoless mutant of
Escherichia coli by carbon 13 nuclear magnetic
resonance. J. BioI. Chem., 258(5), 2906-2910
(1983).
14. Munford, R. S., et al., Sites of tissue binding and
uptake in vivo of bacterial lipopolysaccharide-high density lipoprotein complexes: studies in the rat
and squirrel monkey. J. Clin. Invest., 68(6),
1503-1513 (1981).
15. Morrison, D. C., and Rudbach, J. A., Endotoxin-
cell-membrane interactions leading to
transmembrane signaling. Contemporary Topics
in Molecular Immunology, 8, 187-218, P. Inman
and J. Mandy, eds., Plenum Press, New York
(1981).
16. Galanos, C., et al., International Review of
Biochemistry, Biochemistry of Lipids III, 14, 2309, T. E. Goodwin, ed., University Park Press,
Baltimore (1977).
17. Kurtz, H. J., et al., Effects of continuous
intravenous infusion of Escherichia coli endotoxin into swine. Amer. J. Vet. Res., 43, 262-8 (1982).
18. Rick, P. D., and Young, P. A., Isolation and
characterization of a temperature-nsitive lethal
mutant of Salmonella typhimurium that is
conditionally defective in 3-deoxy-D-manno-
octulosonate-8-phosphate synthesis. J. Bacteriol., 150, 447-55 (1982).
19. Oppenheim, J. J., et al., Immunol. Today, 7, 45
(1986).
20. Skelly, R., et al., Stimulation of T-independent
antibody respons by hapten-lipopolysaccharides without repeating polymeric structure. Infect.
Immun., 23(2), 287-293 (1979).
21. Customer report.
22. Wrenger, E. et al., Peritoneal Mononuclear Cell
Differentiation and Cytokine Production in
Intermittent and Continuous Automated Peritoneal Dialysis. Am. J. Kidney Dis., 31, 234-241 (1998).
23. Hawkins, D. L. et al., Human interleukin 10
suppress production of inflammatory mediators by LPS-stimulated equine periotoneal
macrophages. Vet. Immunol. Immunopathol., 66,
1-10 (1998).
FEB/MES/NSB 7/03
LPS Table
Source organism Extraction method Gel
Filtration Gel Filtration
γ-irr.
Ion-
exchange
Detoxified Gel
Filtration
FITC label
Gel Filtration
TNP label
O26:B6 E. coli Phenol - L8274
TCA – L3755 L2762 L2654 F7037* T7143*
O55:B5 E. coli Phenol - L2880
TCA – L4005 L2637 L6529 L4524 L9023 F8666 T6682
O111:B4 E. coli Phenol – L2630
TCA – L4130 L3012 L4391 L3024 L3023 F3665 T3382
O127:B8 E. coli Phenol – L3129
TCA – L3880 L3137 L4516 L5024 L8654 F3540*
O128:B12 E. coli Phenol – L2755
TCA – L4255 L2887 T6769
E. coli EH-100 (Ra mutant) Ph/Chl/Pet - L9641
E. coli F-583 (Rd mutant) Ph/Chl/Pet – L6893
E. coli J5 (Rc mutant) Ph/Chl/Pet – L5014
L7520*
E. coli K-235 Phenol – L2143
宝石鉴定TCA – L2268 L2018
Klebsiella pneumoniae Phenol – L4268
TCA – L1519 L1770*
Pudomonas aeroginosa 10 Phenol – L9143
TCA – L7018 L8643
Salmonella abortus equi Phenol - L5886
TCA – L6636 L1887
Salmonella enteritidis Phenol - L6011
TCA – L6761 L2012 L7770 L4774 L3773
Salmonella minnesota Phenol - L6261
TCA – L7011 L2137 L4641 L1523* F4665* T3520*
Salmonella minnesota
strain R5
Ph/Chl/Pet - L8893 Salmonella minnesota
曹丕简介
strain R7 (Rd mutant)
Ph/Chl/Pet - L9391
Salmonella minnesota strain Re595 (Re mutant) Ph/Chl/Pet - L9764
L7645*
Salmonella typhimurium Phenol – L6511
TCA - L7261 L2262 L6143 L2525 F4790* T4145*
Salmonella typhimurium
strain SL684 (Rc mutant)
Ph/Chl/Pet - L5891 Salmonella typhimurium
strain SL1181 (Re mutant)
Ph/Chl/Pet - L9516 Salmonella typhimurium
城市发展方向电脑主板品牌
strain TV119 (Ra mutant)
Ph/Chl/Pet - L6016
Salmonella typhosa Phenol - L6386
TCA - L7136
L2387 L7895
F4292*
Serratia marcescens Phenol - L6136
L2512* L4766*
Shigella Flexneri A1 Phenol – L4393
TCA – L7143 L9018
Shigella flexneri (Re mutant) Ph/Chl/Pet - L6643
Vibrio cholerae rotype Inaba 569B Phenol - L0385
F5009* T1271*
* = discontinued product number
Ph/Chl/Pet = phenol:chloroform:petroleum ether
Sigma brand products are sold through Sigma-Aldrich, Inc.
Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchar must determine the suitability of the product(s) for their particular u. Additional terms and conditions may apply. Plea e rever side of
the invoice or packing slip.
