1. 샘플 준비 <ul class="none"><li> 컴파 운드 혼합 1 : Coumarin, 이부 프로펜, 그리고 Carbamazepine, 20 MG / ML 메탄올 각 </li><li> 컴파 운드 혼합 2 : 이부 프로펜과 Carbamazepine, 메탄올의 각 MG / ML 20 </li><li> 컴파 운드 혼합 3 : 이부 프로펜과 Carbamazepine, (디메틸 sulfoxide) DMSO의 각 MG / ML 20 </li><li> 컴파 운드 혼합 4 : 10 MG / ML Fenoprofen, 15 MG / ML 케토프로펜, 20 MG / ML Flavone, 20 MG / ML의 이부 프로펜, 그리고 메탄올 20 MG / ML Coumarin </li></ul> 2. 실험 조건 <ol><li> 분석 크로마 토그래피는 워터스 방법 역 SFC 시스템에서 수행되었다. 칼럼 : Viridis SFC 2 - Ethylpyridine 칼럼, 5 μm의, 4.6 X 150mm, 부품 번호 186004937. </li><li> 예비 크로마 토그래피는 워터스 준비 100 SFC의 UV 감독 시스템에서 수행되었다. 열 : <ul><li> Viridis SFC 2 - Ethylpyridine OBD 컬럼, 5 μm의, 19 X 150mm, 부품 번호 186004945 </li><li> Viridis SFC 실리카 OBD 컬럼, 5 μm의, 19 X 150mm, 부품 번호 186004918 </li><li> Viridis SFC 2 - Ethylpyridine OBD 컬럼, 5 μm의, 19 X 100mm, 부품 번호 186004944 </li></ul></li><li> 다른 방법 매개 변수 : <ul><li> 칼럼 온도 : 40 ° C </li><li> Cosolvent : 메탄올 </li><li> 사출 볼륨, 유량, 그리고 그라디언트가 : 숫자에 보고된 </li></ul></li></ol> 3. 대표 결과 유량 예비 LC 분리에 사용되는 유량은 길이와 직경 열, 입자 크기 및 시스템 backpressure를 포함한 여러 요인에 의해 제한됩니다. 낮은 점도의 이산화탄소, SFC의 모바일 단계의 주요 구성 요소는 일반적으로 액체 크로마 토그래피에 사용되는보다 높은 흐름 속도의 사용이 가능합니다. 열 길이와 연관된 backpressure는 해상도를 높이기 위해 더 열을의 사용을 활성화도 낮습니다. 높은 유량은 분리를 완료하는 데 시간을 줄임으로써 처리량을 향상시킬 수 있습니다. 그림 1에 표시된 바와 같이, 색층 프로필은 열 볼륨 당 퍼센트의 변화로 표현되는 그라디언트의 기울기가 일정하게 유지 제공하는 다른 흐름 속도에서 동일합니다 ... 이것은 유량이 증가와 같은 비율의 실행 시간을 줄임으로써 수행됩니다. <img src="/files/ftp_upload/2604/2604fig1.jpg" alt="그림 1" /> 샘플 해산 및 솔벤트 로딩 혼합물에서 혼합물을 분리하면 샘플 주입하기 전에 완전히 용해되어야합니다. 메탄올, SFC 공동 용매 가장 일반적인, 일반 예비 분리를 위해 일반적으로 요구되는 높은 농도에서 모든 샘플을 solubilize하지 않습니다. 메탄올과 같은 용매의 대형 볼륨은 크로마 토그래피를 왜곡 수있는 강한 용매 효과에 의한 열의 대량 용량을 제한할 수 있습니다. 수정자 흐름 주입, 모든 워터스 준비 SFC 시스템의 표준 특허 구성이 용매 효과를 줄이기 위해 설계되었습니다. 디메틸 sulfoxide (DMSO)는 화합물의 다양한 유형을 solubilize을 정화 연구소에서 정기적으로 사용됩니다. 그림 2, 3 메탄올 주입하는 샘플에 비해 SFC의 DMSO 주사가 achiral 애플 리케이션을위한 높은 로딩과 더 나은 해상도로 발생할 수 방법을 보여줍니다. 참고 : chiral 어플 리케이션의 경우,주의가 DMSO가 심각하게 전통적인 코팅 chiral 정지 단계를 손상시킬 수로 간주되어야합니다. DMSO는 샘플 용해도에 필요한해야하는 경우, 고정 chiral 컬럼이 선택되어야합니다. <img src="/files/ftp_upload/2604/2604fig2.jpg" alt="그림 2" /> <img src="/files/ftp_upload/2604/2604fig3.jpg" alt="그림 3" /> 선택성 SFC에서는 여러 열이 일상적 혼합물의 구성 요소에 대한 최고의 해상도와 피크 모양을 제공하는 확인하기 위해 테스트합니다. 화합물 사이 좋은 해결책은 고순도와 대상 화합물 isolations로 연결됩니다. 아래 예제에서, 2 ethylpyridine 컬럼 샘플 혼합물의 다섯 화합물의 우수한 분리를 보여줍니다. 그것은 다른 선택도를 가지고 있기 때문에 실리카 칼럼에 대한 분석 같은 샘플 혼합물은 세 구성 요소 봉우리를 보여줍니다. 0.8 분 정도하고 약간 이상 flavone과 fenoprofen coelute에서 Coumarin과 이부 프로펜 coelute. Flavone과 이부 프로펜은 또한 실리카 칼럼에서 용리 순서를 변경합니다. 케토프로펜 두 컬럼의 다른 봉우리에서 온 잘 구분합니다. <img src="/files/ftp_upload/2604/2604fig4.jpg" alt="그림 4" /> 스케일링 원유 샘플 혼합물은 일반적으로 유기 용매의 낮은 비율에서 시작하고 비교적 짧은 시간에 유기 용매의 높은 비율로 끝나는 심사 그라디언트로 분석됩니다. 관심의 화합물 잘 해결하는 경우 경사도가 정화 직접 확장할 수 있습니다. 모든 화합물은 일 전에 elute 경우전자 그라디언트의 완료 완벽하게 해결되며, 그라디언트의 길이에 간단한 감소가 허용됩니다. 수정된 그라디언트와 함께 SFC에 사용되는 빠른 흐름 속도가 상당히 목표 화합물의 정화에 필요한 총 시간을 단축. 스케일링 분리는 열 화학 및 입자의 크기뿐만 아니라 적절하게 크기를 조절 주입 볼륨 및 그라디언트와 일치해야합니다. 워터스 예비 SFC 열이 워터스 분석 SFC 컬럼에 우수한 침대 안정성과 유사한 성능을 보장 OBD 기술과 함께 포장됩니다. OBD 예비 열이 밀접하게 상응 분석 컬럼을 일치 밀도 잠자리에 포장됩니다. 이 혁신적인 절차는 우수한 안정성, 재현성, 그리고 효율적으로 예비 열을 생산하고 있습니다. 로딩 연구는 샘플 질량이 칼럼에서 로드할 수있는 얼마나 많은 결정하는 작은 규모에서 수행됩니다. 목표는 해상도를 손상시키지 않고 부하를 극대화하는 것입니다. 향상된 최대 해상도는 격리된 화합물에 대한 높은 부하와 더 순도가 발생합니다. 높은 열 부하는 후속 실험에 대한 충분한 자료를 구하기에 필요한 실행의 수를 줄일 수 있습니다. 로딩 연구에서 크로마 토그래피는 그림 5에 그려져 있습니다. 보수 35 μL 주입 부피는 혼합물에있는 화합물의 모든과 이후에 큰 열의에 예비 실행에 대한 크기를 조정합니다 사이 좋은 해상도를 보여줍니다. 600 μL의 총 볼륨 19 X 150mm 예비 SFC 칼럼에 주입했다. 그림 6은 절연을 위해 사용되는 대규모 크로마 토그래피와 최대 하중에서 소규모 수정 그라데이션을 사용하여 크로마 토그래피를 비교합니다. 절연 및 유지 시간과 시스템 볼륨 및 사출 모드를 포함하여 요인 수있는 분석 및 예비 chromatograms 사이의 해상도로 purification.The 차이에 대해 조사하고 도구로 분석 크로마 토그래피를 사용하는 경우 선택도는 중요한 요소 동일합니다. 특히, 특허 수정자 스트림 분사 모드 *는 용매 효과를 최소화하기 때문에 처리량과 효율성을 향상, 모든 워터스 예비 SFC 시스템에 대한 고용 수 있습니다. * 미국 특허 # 6,576,125 <img src="/files/ftp_upload/2604/2604fig5.jpg" alt="그림 5" /> <img src="/files/ftp_upload/2604/2604fig6.jpg" alt="그림 6" />

<ol>
<li>Optimum Bed Density (OBD) Columns: Enabling Technology for Laboratory-Scale Isolation and Purification. Waters White Paper 720001939EN 2006 Nov.</li>
</ol>

관심 없음 충돌 선언하지 않습니다.

<ul><li> 액체 크로마 토그래피의 기본 원리는 초임계 유체 크로마 토그래피 (SFC)에 적용할 수 있습니다. </li><li> 초임계 유체 크로마 토그래피는 증가 흐름 속도 빠르게 isolations을 만들고 처리량을 증가에서 실행할 수 있습니다. </li><li> 제한된 용해도와 샘플은 디메틸 sulfoxide의 용해와 SFC를 사용하여 정화하실 수 있습니다. 샘플 해산 DMSO를 사용하면 샘플 용해도와 관련된 불확실성을 감소하여 정화 작업이 쉬워집니다. </li><li> 메탄올에 녹아있는 동일한 샘플에 비해 DMSO에 녹아있는 샘플의 해상도와 로딩을 향상시킬 수 있습니다. 더 나은 샘플 해상도 높은 순도와 제품에 연결됩니다. 높은 로딩은 정화 과정의 효율성을 향상시킵니다. </li><li> 열 선택도는 혼합 구성 요소의 분리에 영향을 미칩니다. 빠른 그라디언트 서로 다른 컬럼을 심사하는 것은 예비 칼럼이 예제의 화합물을 분리에 사용할 수있는 대한 결정에 이르게. </li><li> 분석 및 예비 OBD 컬럼 사이 확장할 수있는 능력은 사출 볼륨, 경사 및 기울기 시간이 올바르게 크기 조정 및 화학 및 입자 크기가 일정하게 유지되어있는 크고 작은 비늘 모두에서 동일 분판에 연결됩니다. 열 확장성 따라서 높은 정화 처리 효율을 촉진, 최대 식별의 불확실성을 제거합니다. </li></ul>

<table><tbody><tr><td>Method Station SFC System</td><td>Waters</td><td>More information available at www.waters.com</td><td>Analytical SFC system</td></tr><tr><td>SFC-UV Prep 100</td><td>Waters</td><td>More information available at www.waters.com</td><td>Preparative SFC System</td></tr><tr><td>Viridis&amp;#8482; SFC 2-Ethylpyridine, 4.6 x 150 mm, 5 &amp;mu;m Column</td><td>Waters</td><td>186004937</td><td>Analytical SFC Chromatography Column</td></tr><tr><td>Viridis&amp;#8482; SFC 2-Ethylpyridine OBD&amp;#8482;, 19 x 150 mm, 5 &amp;mu;m Column</td><td>Waters</td><td>186004945</td><td>Preparative SFC Chromatography Column</td></tr><tr><td>Viridis&amp;#8482; SFC Silica OBD&amp;#8482;, 19 x 150 mm, 5 &amp;mu;m Column</td><td>Waters</td><td>186004918</td><td>Preparative SFC Chromatography Column</td></tr><tr><td>Viridis&amp;#8482; SFC 2-Ethylpyridine OBD&amp;#8482;, 19 x 100 mm, 5 &amp;mu;m Column</td><td>Waters</td><td>186004944</td><td>Preparative SFC Chromatography Column</td></tr></tbody></table>

basic principles for purification using supercritical fluid chromatography - advertisement

초임계 유체 크로마 토그래피 (SFC)는 새로운 기술은 아니지만, 예비 SFC는 계측, 소프트웨어 및 화학의 발전과 함께 점점 더 인기를 끌고있다. SFC와 화합물을 분리를위한 기본 원칙은 대규모 예비 액체 크로마 토그래피에 대한 기본 규칙과 비슷합니다. 본 연구에서는, 우리는 유량, 용매 샘플 해산 및 혼합물에서 혼합물의 분리에 대한 열 선택도의 효과를 보여줍니다. 최적 침대 밀도 (OBD) 기술 1과 일치 화학 모여 열 큰 예비 분리에있는 작은 규모에서 간단하고 예측 가능한 확장성을 보장합니다.

초임계 유체 크로마 토그래피 (SFC)를 사용하여 혼합에서 화합물의 분리에 대한 기본 원칙은 예비 액체 크로마 토그래피의 기본과 비슷합니다.

초임계 유체 크로마 토그래피를 사용하여 정제를위한 기본 원칙 - 광고

Although supercritical fluid chromatography (SFC) is not a new technique, preparative SFC is becoming increasingly more popular with advances in ...

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비디오: 초임계 유체 크로마 토그래피를 사용하여 정제를위한 기본 원칙 - 광고

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

울트라 성능 액체 크로마토 그래피 - 질량 분석법 (UPLC-MS)에 의해 혈청 대규모 비 타겟 Metabolomic 프로파일

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

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Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Ultraperformance 액체 크로마토 그래피 고해상도 질량 분석 (UPLC-HRMS)를 사용하여 생물 소스에서 타겟이 불분명 한 대사

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

타겟 안정 동위 원소 희석 액체 크로마 토그래피 - 질량 분광법 분석을위한 세포 지질 추출

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

생체공학

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

신생아 Hypoxia의 생화 학적 측정

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

의학

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

곤충 Antennal 엽에 가스 크로마토 그래피 - 멀티 유닛 레코딩 (GCMR)를 사용하여 강한 휘발성 물질의 식별

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

신경과학

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

씬 크로마 토그래피 (TLC)에 의해 Arabidopsis thaliana 폴라 Glycerolipid의 프로파일은 기체 - 액체 크로마 토그래피 (GLC)와 결합

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

생물학

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

단백질 정제에서 사용하기 위해 자동화된 소수성 상호 작용 크로마 토그래피 칼럼 선정

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104의 정화, 단백질 Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

전자 코를 사용하여 과일 휘발성 분석

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

의 DNA 조각의 분리를위한 아가로 오스 젤 전기 영동

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...