Name: a method for extracting pigments from squid doryteuthis pealeii
Uploaded: 2016-11-09T15:00:00.000+00:00
Duration: 11 min 3 s
Description: Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

미국에서 규제되지 않습니다 여기에 실시 무척추 동물 연구; 따라서 기관 동물 관리 및 사용위원회는 프로토콜의 검토에 대한 권한이 없습니다. 이러한 미국의 규제의 관할에 해당하지 않는 대신에, 저자는 이에이 연구는 진지한 윤리적 사용을 향해 노력, 관리, 및 동물의 치료, 개인의 수를 최소화하고, 이러한 노력으로 수행 하였다 상태 것을 바젤 선언과 실험 동물 과학을위한 국제위원회 (ICLAS) 윤리 지침과 일치한다. 1. Doryteuthis의 pealeii (D.의 pealeii) 해부 <ol><li> 구매 참수 오징어 D. 우즈 홀, MA에있는 해양 생물 연구소의 해양 자원학과 또는 신선한 생선 시장에서 pealeii. 필렛 또는 이전에이 절차를 적용하기 위해 변경 한 시료를 사용하지 마십시오. 구체적으로는, 크로마tophore 층은 여전히 ​​표본에 그대로 있어야합니다. <ol><li> 동물을 즉시 사용하지 않는 경우, 사용할 때까지 -20 ℃에서 보관. 시료를 해동하기 전에 절개 1 시간 동안 실온의 물에 넣습니다. </li></ol></li><li> 동물의 후방 측을 따라 절단 스테인레스 스틸 직선 미세 점 해부 가위를 사용. 복부 맨틀의 기지에서 시작하고, 핀 영역에서 끝난다. </li><li> 집게를 해부 및 해부 가위를 사용하여 결합 조직을 절단로 들어 올려 내부 장기 (위, 아가미, 잉크 선, 생식 기관, 전신 심장과 상완 마음)을 모두 제거합니다. 표본 가방에 장기를 폐기하십시오. </li><li> 표준 11-1 / 2 (지느러미 쪽까지) 맨틀을 핀 T 핀을 해부 사용 &quot;X 7-1 / 2&quot;X 1-1 / 2 &quot;유연한 해부 패드를 포함하는 알루미늄 해부 팬.에 조직을 잠수함 일회용 폴리스티렌 진공 여과 시스템을 통해 여과 된 해수 250 ml의0.2 ㎛의 세공 크기를 포함하는. </li><li> 조심스럽게 그대로 색소 포 포함하는 피부 층을 유지, 해부 포셉 표피 피부 층 (불투명 한 색상)을 제거합니다. 표피층을 제거한 후에 시료 봉투에 버린다. </li><li> 포셉 및 해부 가위로 색소 포 층의 작은 (1cm X 1cm) 섹션을 해부하다. 1.5 ml의 마이크로 원심 분리기 튜브에서 샘플을 수집 (해부 조직과 각 빈 튜브의 절반을 채우기). </li><li> 튜브를 포함하는 조직에 여과 해수의 500 μl를 추가합니다. 샘플을 4 ℃에서 하룻밤 동안 저장 될 수있다. </li></ol> 2. 분리 색소 포 안료 과립 <ol><li> 조직 소화 <ol><li> 파파인 / 콜라게나 제의 제조 <ol><li> 종이의 무게 콜라게나 제 30 mg을하고, 4 &quot;× 4&quot;낮은 질소를 사용하여 파파인 5 ㎎을 무게. 스크류 캡으로 50 ML의 폴리 프로필렌 원심 분리 관에 다음을 전송합니다. </li><li> deioniz 10 ㎖를 측정눈금 피펫을 사용하여 에드 물. 파파인 / 콜라게나 제를 포함하는 유리 병에 직접 추가 할 수 있습니다. 솔루션까지 가장 높은 설정에 소용돌이는 분명하다. </li></ol></li><li> 세포 외 조직을 제거 <ol><li> g X 14000에서 5 분 동안 단계 170에서 수집 된 색소 포 조직을 원심 분리기. 플라스틱 폐기물 통에 바닷물의 상단 층을 폐기하십시오. </li><li> P1000 가변 볼륨 단일 채널 마이크로 피펫을 사용하여 조직에 파파인 / 콜라겐 용액 500 μl를 추가합니다. 가장 높은 설정에 1-2 분 동안 각각의 샘플을 소용돌이. </li><li> 주변 조직으로부터 세포를 해리 40 kHz의 주파수에서 5 분 동안 샘플을 초음파 처리. </li><li> 14,000 × g에서 5 분 동안 원심 분리기. 플라스틱 폐기물 통에 상층 액을 버린다. </li><li> g X 14000에서 5 분 동안 수집 된 색소 포 조직을 원심 분리기. 플라스틱 폐기물 통에 바닷물의 상단 층을 폐기하십시오. 다시 한 번 반복합니다. </li><li> 수동으로 남아있는 큰 조직 초를 제거다음 단계로 진행하기 전에 포셉이 과정에서 소화되지 않은 TIONS. </li></ol></li></ol></li><li> 안료 과립을 분리 <ol><li> 균질화 버퍼의 준비 <ol><li> 2.38 g의 무게 (4- (2- 히드 록시 에틸) -1- 피페 라진 에탄 술폰산) (HEPES, 100 mM)을 염화 마그네슘 (10 mM)을, 칼륨 아스파 테이트의 0.856 g (50 mM)을 0.095 g, 0.015 g의 디티 오 트레이 톨 (1 mm)이다. 스크류 캡을 가진 150 ㎖ 폴리스티렌 용기에 옮긴다. 하나는 상용 미니 태블릿 프로테아제 억제제를 추가합니다. </li><li> 눈금 실린더를 사용하여 100 ml의 탈 이온수를 측정한다. 균질화 염을 함유하는 용기에 직접적으로 추가한다. 솔루션까지 소용돌이는 분명하다. </li></ol></li><li> 안료 과립의 균질화 <ol><li> 5 분 동안 14,000 XG에서 2.1 단계에서 각 샘플을 원심 분리기. 플라스틱 폐기물 통에 상층 액을 버린다. 이 샘플은 세포 조직의 무효합니다. </li><li> 추가 500# 181; 1-2 분마다 각각의 튜브와 소용돌이에 균질화 버퍼의 난 철저하게 샘플을 혼합합니다. 30 분 (~ 40 kHz에서)에 대한 샘플을 초음파 처리하고 14,000 × g에서 원심 분리의 5 분에 따릅니다. </li><li> 플라스틱 폐기물 통에 뜨는을 폐기하고, 색소 포의 sacculus 단백질 사슬의 완전한 소화를 보장하기 위해 이전 단계를 2 ~ 3 번 반복합니다. 남은 용액은 담황색 상청과 분리 된 안료 입자를 함유하는 붉은 색의 펠렛을 함유한다. </li></ol></li></ol></li></ol> 3. 안료 추출 <ol><li> 산성 메탄올의 제조 (염산 - 메탄올) <ol><li> 조심스럽게 메탄올 5 ㎖ (메탄올)로 농축 (36.5-38 % (/ w) w) 염산 (HCL)의 25 μl를 추가합니다. 스크류 캡 유리 샘플 유리 병에 염산 - 메탄올 용액을 저장합니다. 균일 용액을 섞어 부드럽게 소용돌이. 주 : 더 산성 용액 더 안료는 인터넷 동안 추출한다첫 번째 추출. </li></ol></li><li> 안료의 추출 <ol><li> 14,000 XG에서 5 분 (2.2.2)에서 색소 과립 현탁액을 원심 분리기 및 플라스틱 쓰레기 통에 상층 액을 버린다. </li><li> HCL에 - 메탄올 용액 500 μl를 추가하고 3-4 분 ~ 각 샘플을 소용돌이. 10 분 (~ 40 kHz에서)를위한 샘플을 초음파 처리 한 후 14,000 × g에서 5 분간 원심 분리. </li><li> 전송 피펫을 사용하여, 1 DRAM 나사 최고 유리 병에 뜨는를 수집합니다. 상층 액은 색상 진한 빨간색이어야한다. </li><li> 더 색 (~ 4-5 세척) 추출되지 않을 때까지 추출 단계 3.2.2를 반복합니다. 참고 : 각 추출 ~ 안료의 1.5 ml를 얻을 것입니다. 남아있는 무색의 펠릿 안료 추출 과립이다. 주 : 무색 과립 추출 안료 모두 더 사용할 때까지 39 ° C의 물에 저장 될 수있다. </li></ol></li></ol> 추출 과정에서 4. 특성 <ol><li> 스캔 엘ectron 현미경 <ol><li> 추출 처리의 순도를 확인하기 위해 주사 전자 현미경 (SEM)을 사용한다 (단계 2.2.1 안료 추출 단계 3.2.3부터 미 반응 된) 이미지 과립. </li><li> 14,000 XG에서 5 분 동안 영상, 최초의 원심 분리기 모두 반응 과립 및 안료 추출 과립을위한 과립 샘플을 준비하고 플라스틱 폐기물 통에 뜨는을 취소합니다. 1 ml의 에탄올 및 예금 전송 피펫을 사용하여 알루미늄 SEM 스텁 상 과립 현탁액을 2 ~ 3 방울에이를 다시 일시 중지합니다. </li><li> 밤새 샘플을 건조시킨 후, 15 nm의 코팅을 얻기 위해 3 분 동안 금 - 백금 코팅 피막 스퍼터. </li><li> 쇼트 키 방출하고 2 차 전자 검출기와 SEM을 사용하여 이미지. 5-7 kV의 광 전압 및 이미지 8 9mm 이들 물질 사이의 작동 거리를 사용한다. </li></ol></li><li> 자외선 - 가시 광선 (UV-힘) 분광 측정 <ol><li> 흡광도 프로 모니터200-800 nm의에서 듀얼 빔 분광 광도계를 사용하여 3 조건 파일 (2.2.2에서 미 반응 과립, 3.2.3에서 추출 색소 및 3.2.4에서 색소를 추출 과립). </li><li> 미 반응 과립 및 안료 추출 과립 1 ml의 큐벳에 탈 이온수를 사용하여 빈 측정 값을 수집합니다. 두 샘플에 대한 UV-마주 흡수 스펙트럼을 수집합니다. </li><li> 추출 된 안료 1 mL를 큐벳에서 산성 메탄올 용액을 사용하여 블랭크 측정을 수집한다. 샘플에 대한 UV-마주 흡수 스펙트럼을 수집합니다. </li><li> 흡광도 피크의 최대 상대 높이에 도달하는 파장을 식별함으로써, 각 시료의 최대 파장을 결정한다. </li></ol></li></ol>

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우리는 오징어 색소 포 과립에서 색소를 추출하는 방법을 설명했다. 특히 과립을 대상으로, 우리의 목표는 적응 착색을 매개의 역할을 결정하는 것입니다. 이 방법은 대량 조직 샘플 (14) 또는 동결 건조 피부 (15)를 사용하여 두족류 안료의 특성을 설계 이전 보고서에서 다르다. 이 프로토콜은 색소 포 안료를 추출하는 데 효과적이지만, 이것은 작은 반응 규모...

오징어, 오징어, 문어와 같은 두족류는 동적으로 위장 및 신호에 대한 자신의 모양을 변경할 수있는 기능을 가지고있다. 1-6이 기능은 색소로 알려진 착색 기관의 선택 면적 확장에 의해 부분적으로 지원됩니다. 4,7-9 색소는 이들이 작동되면 근섬유로 방사상 고정되는 cytoelastic sacculus 내에 한정 나노 안료 입자의 네트워크를 포함 소프트 액츄에이터. 1,3, 색소는 장기에 걸쳐 입자를 분산되게 표면적을 500 %로 확대. 3,7-을 이 작업은 색소 세포의 수 이상으로 행한 경우, 10, 11는, 동물의 전체 착색이 변경됩니다. 이 색소 과립이 색의 변화에 ​​기여하는 것으로 알려져 있지만, 이들 조성물은 알려지지 않았다. 우리는 분리 및 미래 연구를 조성하도록 구성 될 수있다 안료 색소 포를 정제하는 방법을 설명한다. 3,12 색소 포 함유 조직은 두족류에서 조심 근절을 통해 수거된다. 소화 균질화 과정은 다음 색소 포 세포 주변 조직을 해리와 구분하기 위해 사용된다. 나노 구조 과립 반복 초음파 처리 및 원심 분리를 이용하여 남아있는 색소로부터 단리하고 정제한다. 정제시, 안료, 산성 메탄올 용액을 사용하여 나비 날개에서 보이는 색상의 추출에서 적응하는 과정에서 입자로부터 추출된다. (13) 주사 전자 현미경 (SEM) 및 측정법은 색소 포 안료가 성공적으로 사용하여 추출하는 것을 확인하기 위해 사용 이 과정. 이 방법은 C의 분자 기여를 탐구하는 데 사용되는 색소 포 과립의 분리를 설명oloration 두족류에. 전체 동물의 12 작은 분자 추출은 종종 길고 지루한 과정이 될 수 있습니다. 여기에 목표는 두족류의 나노 입자의 안료의 취득을위한 효과적이고 손쉬운 프로토콜의 미래 연구원을 알려드립니다.

<table><tbody><tr><td>Collagenase</td><td>Alfa Aesar</td><td>9001-12-1</td><td>No hazard</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3482-12-3</td><td>Irritant, acute toxicity</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td>Mild irritant</td></tr><tr><td>Hydrochloric acid</td><td>EMD Chemicals</td><td>7647-01-0</td><td>Corrosive</td></tr><tr><td>k-Aspartate</td><td>Sigma-Aldrich</td><td>1115-63-5</td><td>Reacts violently with oxidants</td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Mild eye irritant</td></tr><tr><td>Methanol</td><td>Pharmco-AAPER</td><td>67-56-1</td><td>Highly flammable</td></tr><tr><td>Mini tablet prtoease inhibitor</td><td>Sigma-Aldrich</td><td>469315-90-01</td><td>Corrosive to metal and skin</td></tr><tr><td>Papain</td><td>Sigma-Aldrich</td><td>9001-73-4</td><td>Irritant&amp;nbsp;</td></tr></tbody></table>

a method for extracting pigments from squid doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

색소는 D.에서 해부하는 pealeii 등의 맨틀 (그림 1A, 1B). 그들은이 제거되면 색소 세포가 용해 및 색소 과립 (도 2A, 2B)를 밖으로 분리하는 원심 분리 및 세척주기를 사용하여 정제한다. 산성 메탄올 용액 (염산 - 메탄올)는 수용성 색소 추출물과 불용성 무색 안료 추출 과립을 산출, 과립 (그림 2C)에서</strong...

Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

A Method for Extracting Pigments from Squid Doryteuthis pealeii

오징어 Doryteuthis pealeii 색소의 나노 입자에서 색소의 추출을위한 프로토콜이 표시됩니다.

오징어로부터 색소를 추출하는 방법<em&gt; Doryteuthis의 pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

a-method-for-extracting-pigments-from-squid-doryteuthis-pealeii

Research

JoVE Journal

Biology

A Method for Extracting Pigments from Squid Doryteuthis pealeii

9.3K 조회수.  University of New Hampshire.  오징어 Doryteuthis pealeii 색소의 나노 입자에서 색소의 추출을위한 프로토콜이 표시됩니다. 

비디오: A Method for Extracting Pigments from Squid Doryteuthis pealeii

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

물고기 먹이 실험 생물 검정은 해양 생물의 조직에서 이차 대사 산물의 Antipredatory 활동을 평가하기 위해

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

연구

환경과학

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

복부 색소 침착 정량화<em&gt; Drosophila melanogaster</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

유전학

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

오일 유제에서 물 : 소수성 안료와 수용성 엽록소 결합 단백질을 조립하기위한 새로운 시스템

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

화학

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

분리 및 통합 규조류 Cyclotella Meneghiniana Thylakoid 지질으로 리에서에서 광 수확 안테나

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

생화학

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a large membrane protein complex coordinating hundreds of light harvesting and electron transport factors and is the most efficient light harvesting system found in nature. Photons absorbed by the four LHCA antenna proteins that make up LHCI are transferred through excitonic interaction to the PSI core reaction center and are used to facilitate light-driven charge separation across the thylakoid membrane, providing reducing power and energy for carbon fixation in photoautotrophic organisms. The high quantum efficiency of PSI makes this complex an excellent model to study light-driven energy transfer. In this protocol, plant tissue is mechanically homogenized, and the chloroplasts are separated from the bulk cellular debris by filtration and centrifugation. The isolated chloroplasts are then osmotically lysed, and the thylakoid membranes are recovered via centrifugation and solubilized using the detergent n-dodecyl-beta-maltoside. The solubilized material is loaded onto an anion exchange column to collect most of the chlorophyll-containing complexes. Larger complexes are precipitated from the solution, resuspended in a small volume, and loaded on sucrose gradients to separate the major chlorophyll-containing complexes. The resulting sucrose gradient fractions are characterized to identify the band of interest containing PSI-LHCI. This protocol is highly similar to the protocol used in the crystallization of plant PSI-LHCI with some simplifications and relies on methods developed over the years in the lab of Nathan Nelson.

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

성적 증명서와 Metabolites에서의 정화<em&gt; Drosophila</em&gt; 헤드

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

생물학

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

식물과 녹조류의 빛 수확 단지의 체외의 재구성에

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

준비 번데기 기간 및 초파리 guttifera 의 날개 색소 측정 방법

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Zebrafish를 사용하여 색소 침착 조절자를 식별하기 위한 역 유전적 접근

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

오징어로부터 색소를 추출하는 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

물고기 먹이 실험 생물 검정은 해양 생물의 조직에서 이차 대사 산물의 Antipredatory 활동을 평가하기 위해

복부 색소 침착 정량화

오일 유제에서 물 : 소수성 안료와 수용성 엽록소 결합 단백질을 조립하기위한 새로운 시스템

분리 및 통합 규조류 Cyclotella Meneghiniana Thylakoid 지질으로 리에서에서 광 수확 안테나

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

성적 증명서와 Metabolites에서의 정화

식물과 녹조류의 빛 수확 단지의 체외의 재구성에

준비 번데기 기간 및 초파리 guttifera 의 날개 색소 측정 방법

Zebrafish를 사용하여 색소 침착 조절자를 식별하기 위한 역 유전적 접근