이 작품은 건강과 낭성 섬유증 재단의 국립 연구소에서 교부금에 의해 지원되었다.

1. LPS 추출을위한 박테리아의 작성 <ol><li> 항생제 필요한 경우에 보충 Luria 국물 (LB), 5 ML에서 하룻밤 문화를 시작합니다. 37시 배양기를 흔들어 ° C ~ 200 RPM의 (12-18시간) 야간 문화를 성장. </li><li> LB와 문화 1시 10분을 희석하고 분광 광도계에서 OD 600 독서 가져가라. OD 600 독서를 바탕으로 0.5의 OD 600으로하여 박테리아의 1.5 ML 서스펜션을합니다. </li><li> 펠렛은 10 분 10,600 X g에서 microcentrifuge의 박테리아. 표면에 뜨는를 제거하고 폐기. LPS를 즉시 추출되지 않을 경우 펠렛은 -20 ° C에 저장할 수 있습니다. </li></ol> 2. LPS의 추출 <ol><li> 첫째, 2X SDS 버퍼를 준비합니다. 0.1에서 50 ML의 4% β-메르 캅 토 에탄올 (BME) 4 % SDS의 솔루션 및 20 % 글리세롤 만들기 M 트리스-HCL, 산도 6.8. 솔루션을 염색하는 bromophenol 파란색의 핀치를 추가합니다. 2X을 diluting하여 1X SDS-버퍼 주식을 만들기멸균 증류수 H 2 O에 관련 째깍 1시 1분 이것은 상온에서 저장할 수 있습니다. </li><li> 멸균 증류수 H 2 O에에 DNase 나 RNase와 Proteinase K의 세 별도의 10 밀리그램 / ML 솔루션 만들기 </li><li> 1X SDS-버퍼 200 μl의 단계 1.3에서 pelleted 박테리아를 Resuspend. 펠렛 완전히 천천히 아래로 솔루션을 pipetting 및하여 resuspended되었는지 확인하십시오. 와동하지 마십시오. </li><li> 15 분 동안 물을 욕조에 정지 박테리아를 끓이다. 15 분 동안 상온에서 냉각 솔루션을 허용합니다. </li><li> 2.2에서 준비 DNase I 및 RNase 솔루션 모두 5 μl를 추가합니다. 30 분간 37 ° C에서 샘플을 품어. *이 단계는 선택 사항입니다, nuclease-대우와 비 대우 샘플 사이 LPS의 품질에 최소한의 차이가있다. </li><li> 2.2에서 준비 Proteinase K의 솔루션을 10 μl를 추가합니다. 3 시간 동안 59 ° C에서 샘플을 품어. 시간 제약이있는 경우이 단계는 하룻밤 사이에 수행할 수 있습니다. </li><li> 각 샘플, 광고까지의 D 200 μl는 얼음처럼 차가운 트리스 - 포화 페놀 (트리스 - 포화 페놀를 사용할 수없는 경우에는 수상 포화 페놀은 대신 사용될 수 있습니다.) 튜브가 꽉 닫혀있에 모자, 약 5~10초위한 와동 각 샘플을 확인합니다. </li><li> 가끔 vortexing, 15 분 동안 65 ° C에서 샘플을 품어. 실온에 시원한 잠복기 후, 다음 1 각 샘플로 룸 온도 디에틸 에테르 ML 5로 10 초간 소용돌이를 추가합니다. 그것이 휘발성이기 때문에, 연기 후드에 핸들 에테르를 수행하십시오. </li><li> 10 분 동안 20,600 X g에서 샘플을 원심 분리기. 조심스럽게 원심 분리기에서 샘플을 제거하고 아래쪽에 파란색 레이어를 추출합니다. 상위, 투명 레이어를 사용하지 않아야합니다. 파란 레이어의 소량 뒤에두면 상위 레이어와 오염 것이 바람직합니다. </li><li> 2.9 - 단계 2.7을 반복하여 샘플을 다시 압축을 풉니다. 두 extractions은 일반적으로 충분합니다. 샘플 흐린 나타나는 경우 자세한 extractions는 performe 수 있습니다라. SDS-PAGE로 분리되기 전에 추출된 표본 각 2X SDS-버퍼 200 μl를 추가합니다. 샘플은 8% -15 %는 SDS-polyacrylamide의 젤에서 실행할 수 있습니다. 이 방법을 사용 준비가 LPS의 다섯 데 15 μl는 시각화를 위해 일반적으로 충분합니다. </li></ol> 3. 대표 결과 위와 같이 준비 LPS 샘플 표준 실버 염색법 프로토콜이나 상용 LPS의 염색법 키트를 사용하여 SDS-PAGE에서 분리에 따라 직접 염색법에 의한 시각이 될 수 있습니다. 또는 polyacrylamide 젤에 분리된 LPS는 nitrocellulose 막에게 양도 및 LPS 특정 안티 세라를 사용하여 immunoblotting 서양를 받게됩니다. 이 프로토콜의 경우, 우리는 프로-Q 에메랄드 300 Lipopolysaccharide 젤 얼룩 키트 (분자 프로브)를 사용하고, 제조 업체의 지침을 따라갔다. 프로-Q 에메랄드 300 스테인드 12% SDS-polyacrylamide 겔은 그림 1에 표시됩니다. 각 레인은 DIF에서 준비 LPS의 15 μl를 포함Burkholderia dolosa의 ferent 변종은 낭성 섬유증 환자의 가래 샘플로부터 격리. 사다리 패턴을 banding 다른 LPS, 다른 숫자 올리 고당 코어에 연결된 장치를 반복 O-항원의 반사,이 방법을 사용하여 증거들이, 예를 들어, 레인 1과 6의 샘플이 서로 유사한 banding 패턴을 가지고 (빨간색 박스). <img alt="그림 1" src="/files/ftp_upload/3916/3916fig1.jpg" /> Burkholderia dolosa 임상 분리의 그림 1. 프로-Q 에메랄드 300 스테인드 LPS. 일곱 B.로부터 LPS 이 프로토콜의 설명에 따라 낭성 섬유증 환자의 확산에서 dolosa의 종자는 고립되었다. 열 다섯 μl는 제조 업체의 지침에 따라, 프로-Q 에메랄드 300과 12% SDS-polyacrylamide 젤, 그리고 스테인드에서 분리되었다. LPS 코어는 화살표를 표시하고, O-항원 반복 유사한 banding 패턴을 보여주는 LPS는 빨간색으로 박스가 있습니다. M은 = 분자량 마커.

<ol>
	<li>Raetz, C. R., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+endotoxins.">Lipopolysaccharide endotoxins.</a> Annu. Rev. Biochem. 71, 635-700 (2002).</li><li>Valvano, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Export+of+O-specific+lipopolysaccharide.">Export of O-specific lipopolysaccharide.</a> Front. Biosci. 8, 452-471 (2003).</li><li>Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide:+a+major+virulence+factor,+initiator+of+inflammation+and+target+for+effective+immunity.">Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity.</a> Int. J. Med. Microbiol. 297, 277-295 (2007).</li><li>Freudenberg, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipopolysaccharide+susceptibility+in+the+innate+immune+response+to+Salmonella+typhimurium+infection:+LPS,+a+primary+target+for+recognition+of+Gram-negative+bacteria.">Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.</a> Microbes Infect. 3, 1213-1222 (2001).</li><li>Jann, K., Jann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+antigens+of+Escherichia+coli.">Polysaccharide antigens of Escherichia coli.</a> Rev. Infect. Dis. 9, S517-S526 (1987).</li><li>McCallum, K. L., Schoenhals, G., Laakso, D., Clarke, B., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-molecular-weight+fraction+of+smooth+lipopolysaccharide+in+Klebsiella+serotype+O1:K20+contains+a+unique+O-antigen+epitope+and+determines+resistance+to+nonspecific+serum+killing.">A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.</a> Infect. Immun. 57, 3816-3822 (1989).</li><li>Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+isolates+from+patients+with+cystic+fibrosis:+a+class+of+serum-sensitive,+nontypable+strains+deficient+in+lipopolysaccharide+O+side+chains.">Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains.</a> Infect. Immun. 42, 170-177 (1983).</li><li>Gupta, S. K., Masinick, S., Garrett, M., Hazlett, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide+binds+galectin-3+and+other+human+corneal+epithelial+proteins.">Pseudomonas aeruginosa lipopolysaccharide binds galectin-3 and other human corneal epithelial proteins.</a> Infect. Immun. 65, 2747-2753 (1997).</li><li>Hitchcock, P. J., Brown, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+heterogeneity+among+Salmonella+lipopolysaccharide+chemotypes+in+silver-stained+polyacrylamide+gels.">Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.</a> J. Bacteriol. 154, 269-277 (1983).</li><li>Westphal, O., Jann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lipopolysaccharides.">Bacterial lipopolysaccharides.</a> Methods Carbohyr. Chem. 5, 83 (1965).</li><li>Lieberman, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+bacterial+evolution+within+multiple+patients+ties+novel+genes+to+pathogenesis.">Parallel bacterial evolution within multiple patients ties novel genes to pathogenesis.</a> Nat Genet. , (2011).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

우리는 핵산과 단백질 등 다른 세포 구성 요소에서 LPS를 멀리 정화하는 방법을 설명했습니다. 이 방법은 그림 1에 표시된 SDS-PAGE의 젤의 탄수화물의 염색법을 포함한 다양한 시각화 방법, 다수에서 사용할 수있는 고품질의 LPS를 제공합니다. 이 방법은 특정 안티 세라를 사용하여 종자의 다양한 LPS를 혈청형하거나 직접 시각화하여 분리 사이 relatedness을 표시하는 데 사용할 수 있습니다. 예를 들어,...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DNase 나 재조합, RNase가없는 </td><td> 로슈 </td><td> 04716728001 </td></tr><tr><td> RNase </td><td> 로슈 </td><td> 10109169001 </td></tr><tr><td> Proteinase K </td><td> 어부 </td><td> BP1700 </td></tr><tr><td> 페놀 트리스 - 포화 </td><td> 어부 </td><td> BP1750-100 </td></tr><tr><td> 디에틸 에테르 </td><td> 토마스 과학 </td><td> C313K31 </td></tr><tr><td> 프로-Q 에메랄드 300 Lipopolysaccharide 젤 얼룩 키트 </td><td> 분자 프로브 </td><td> P20495 </td></tr></tbody></table>

purification and visualization of lipopolysaccharide from gram-negative bacteria by hot aqueous-phenol extraction

Lipopolysaccharide가 (LPS) 그람 음성 세균의 외부 세포막의 주요 구성 요소입니다. 그것은 지방질로 구성된 삼자 간의 분자는 외부 세포막에 박혀있는, 셀 1, 2의 표면에서 바깥쪽을 확장하는 핵심 올리 고당 및 반복 O-항원 단위. LPS는 녹농균, 살모넬라균 종, 그리고 대장균 3-5 및 LPS O-항원 구성 양식 종자의 serotyping의 기초의 차이 등 많은 세균 종의 독성 및 pathogenesis에 중요 immunodominant 분자이다. LPS는 감염의 개시에 세포를 호스트 첨부에 참여하고 보완 - 중재 죽이는에서 보호를 제공합니다; LPS 부족 변종은 독성 6-8에 대한 감쇠가 될 수 있습니다. 이러한 이유로, 그것은 특히 임상 분리에서 LPS를 시각화하는 것이 중요합니다. 특정 의해 패턴과 인식을 banding 떠올리 LPSntibodies는 스트레인 lineages를 식별할 수 있도록, 다양한 돌연변이를 특성화하는 데 유용한 도구가 될 수 있습니다. 이 보고서에서 우리는 그람 음성 박테리아 세포에서 LPS의 분리 및 정제를위한 고온 수성 - 페놀 방법을 설명합니다. 이 프로토콜은 멀리 구 짧고 덜 집약적인 추출 방법으로 발생 LPS의 시각화를 방해할 수있는 핵산과 단백질에서 LPS의 추출을 허용합니다. LPS이 방법은 탄수화물 / 당단백질 얼룩 또는 표준 실버 염색법 방법을 사용하여 나트륨 dodecyl 황산 (SDS)-polyacrylamide 겔 전기 영동 (PAGE)와 직접 스테인드으로 구분됩니다 준비. LPS로 방지 세라 많은 저해 수있는 외부 막 단백질이나 다른 antigenic 목표와 교차 반응 반응은 SDS-PAGE로 구분 원유 세포 lysates의 서양 immunoblot에 따라 관찰되는 항체가 포함되어 있습니다. 혼자 원유 세포 lysates의 단백 분해 효소 처리는 항상 이것을 제거하는 효과적인 방법 없습니다이 문서나 다른 시각화 방법을 사용하여 배경. 또한, 이러한 배경을 제거하기 위해 광범위한 단백 분해 효소 처리가 잘 앞서 언급한 방법 중 하나가 해결되지 품질의 LPS로 이어질 수 있습니다. 이러한 이유로, 우리는 Westpahl 및 Jann 10 일부터 맞게 다음과 같은 프로토콜, LPS 추출에 이상적이라고 생각합니다.

Watch this Scientific Journal Video about Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction at JoVE.com

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

우리는 그람 음성 세균의 lipopolysaccharide을 (LPS) 정화를위한 수정된 뜨거운 수성 - 페놀 추출 방법을 설명합니다. 일단 압축을 푼, LPS는 연속적으로 SDS-PAGE에 의해 분석 및 직접 염색법이나 서양 immunoblot에 의해 시각하실 수 있습니다.

핫 수성 - 페놀 추출에 의한 그람 음성 박테리아에서 정화와 Lipopolysaccharide의 시각화

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

purification-visualization-lipopolysaccharide-from-gram-negative

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

35.0K 조회수.  University of Virginia Health System. 우리는 그람 음성 세균의 lipopolysaccharide을 (LPS) 정화를위한 수정된 뜨거운 수성 - 페놀 추출 방법을 설명합니다. 일단 압축을 푼, LPS는 연속적으로 SDS-PAGE에 의해 분석 및 직접 염색법이나 서양 immunoblot에 의해 시각하실 수 있습니다.

비디오: Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

그람 음성 박테리아에서 분리 및 지질의 화학 특성

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

연구

화학

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

초성능 액체 크로마토그래피에 의한 조성 분석을 위한 세균세포벽의 격리 및 준비

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

추출 및 토양의 미생물 인지질 지방산의 분석

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

환경과학

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

Slp1 중합체 및 단층의 AU-상호 작용<em&gt; Lysinibacillus 스패 리 쿠스</em&gt; JG-B53 - QCM-D, 생체 분자 - 금속 연구 도구로서 ICP-MS와 AFM

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

세균성 병원체 지방산 을 막 인지질로 통합하는 연구를 위해 닭 계란 노른자에서 지단백질 입자의 분리

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

생화학

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

액체 크로마토그래피, 질량 분석법 및 생물정보학에 의한 펩티도글리칸의 반정량 분석

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Ganglioside 추출, 정제 및 프로파일링

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

세균성 단백질 질량 분석에 의하여 구조 결심에 대 한 N 맨끝 Lipopeptides의 준비의 농축

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

면역학 및 감염병학

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

아시네토박터 바우만니에 대한 기술적 조정을 통해 그람 음성 박테리아에 대한 셀 봉투를 내부 및 외부 멤브레인 분획으로 분리

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

얇은 층 크로마토그래피에 의한 진균의 지질 조성 분석

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...