프로토콜은 이전 게시11에서 채택 되었다. T. Z., D. ch.와 J. Č. 의해 지원 되었다 교육, 젊음 및 스포츠 국가 지속 가능성 프로그램 내에서 체코의 난 (NPU 나), 번호 LO1415를 부여. J. Č. 또한 지원 했다가 CR, 부여 번호 18-24397S. 악기 및 기타 시설에 대 한 액세스는 시스템 생물학 C4SYS (아무 LM2015055 프로젝트)에 대 한 체코 연구 인프라에 의해 지원 되었다. M. A. S. [no. 14-14-00904] 러시아 과학 재단에서 교부 금에 의해 지원 되었다.

1. 남조류 재배

<ol><li>Synechocystis 셀 삼각 플라스 크 또는 photobioreactors10,19 (예를 들어, 17 mM HEPES10를 사용 하 여) < 10의 pH를 유지 하기 위해 버퍼 BG11 중간20 에서 육성. 참고: 표준 재배 조건 필요 제어 온도 (일반적으로 30 ℃, 최적 온도 35 ° C)21, 조명 (800 µmol [광양] 최대 강도의 일반적으로, 하얀 빛 / [m2·s])21, 그리고 CO 2 공급 (400 mL 평면 패널 photobioreactor CO2 농도 포화 성장 1700 ppm 이다)21.</li>
	<li>문화의 밀도 확인 하려면 spectrophotometrically 730에서 문화 광 밀도 측정 nm (OD730) 또는 빛 경로 1 cm.의 베트를 사용 하 여 현미경으로 hemocytometer 또는 자동화 된 셀을 사용 하 여 셀의 개수 카운터입니다.</li>
</ol>2. 샘플 준비
<ol><li>Phycobiliprotein 저하를 방지 하기 위해 낮은 irradiance에서 동작.</li>
	<li>안전 잠금 관에 문화 현 탁 액의 3 x 1 mL를 수확. Triplicates를 사용 하 여 측정에서 기술적 오류 추정에 대 한. 무 균 조건 하에서 문화 샘플링을 수행 하려면 층 류 후드에 세포를 수확 하 고 적절 한 작업 및 안전 관행에 따라.</li>
	<li>15000 x g 에 5 분 제대로 균형된 원심 분리기로 터에 관심을 지불에 대 한 실험실 온도에서 세포 원심 원심, 후는 supernatants 삭제 합니다. 수는 펠 렛을 방해 하지 않도록 주의 하십시오.</li>
	<li>냉동 실에 샘플을 넣어. 장기 저장을 위한-80 ° c.에 샘플을 계속 이것은 phycobiliprotein 저하를 방지 해야 합니다. 단기 저장을 위해,-20 ° C 충분 하다.</li>
	<li>Freeze-dry 샘플 하룻밤. 적절 한 동결 과정에 대 한 동결 건조 기 콘덴서-60 ° c의 온도 유지 하 고 동결 건조 기에서 압력 챔버 1 hPa.</li>
	<li>마친 후 동결 주기, 튜브 공기에서 물의 재흡수를 방지 하기 위해 가능한 한 빨리 닫습니다.</li>
</ol>3. 세포 균질 및 추출 색소
<ol><li>각 샘플 튜브 (직경 2 mm)와 유리 구슬의 4 조각을 추가 하 고 튜브를 닫습니다. 참고: 때 균질 사용 안전 잠금 튜브의 뚜껑은 너무 얇은, 그것은 깨뜨릴 수 있는 동안 균질; 따라서, 강한 뚜껑만 안전 잠금 튜브는이 프로토콜에 대 한 것이 좋습니다.</li>
	<li>15 유리 구슬과 샘플 균질 실험실 온도에서 균질 화기에 s. 참고: 제대로 무 균된 샘플 안전 잠금 튜브의 전체 내부 표면에 전염 됩니다.</li>
	<li>1 mL PBS 버퍼 (pH 7.4)의 phycobiliproteins을 추출 하기 위해 샘플을 4 ° C에 precooled를 추가 합니다. 참고:이 단계에서 추출 된 단백질의 저하를 방지 하기 위해 얼음에 샘플 계속. 이것은 중요 한입니다.</li>
	<li>5 샘플 PBS 믹스 실험실 온도에서 균질 화기에 s. 참고: 혼합 후, 샘플은 녹색.</li>
	<li>혼합 후, 샘플 60 분 커버에 대 한 얼음에 색소 저하를 방지 하기 위해 뚜껑 얼음 목욕 유지.</li>
	<li>Phycobiliprotein 추출의 60 분, 후 5 분 원심 분리기 회전자를 제대로 균형에 관심을 지불에 대 한 4 ° C에서 15000 x g 에서 샘플 원심. 참고: 원심, 후에 상쾌한 시안색 파란색이 있다.</li>
</ol>4. Phycobiliprotein 정량화
<ol><li>Spectrophotometric 측정 하기 전에 빈으로 PBS 버퍼를 사용 하 여 기준선을 분 광 광도 계를 보정.</li>
	<li>분 광 광도 계 보정은 일단 무시 한 분 광 광도 계 베트에서 PBS 고 폐기 버퍼 대신 추출된 phycobiliproteins와 상쾌한 플라스틱.</li>
	<li>Phycobiliprotein 농도 spectrophotometrically, 0.5의 슬릿 폭을 사용 하 여 계량 nm.<ol>
<li>Phycocyanin와 615에서 빈 PBS 버퍼에 대 한 allophycocyanin phycocyanobilins의 흡 광도 측정 (615) 및 652 nm (652), 각각, 720에 세포질 파편의 흡 광도 측정 하 고 nm (720).</li>
		<li>Phycocyanin와 방정식에 따르면 allophycocyanin의 (1) 및 (2) 베넷과 Bogorad12의 농도 계산. <img alt="Equation 1" src="/files/ftp_upload/58076/58076eq1.jpg"> <img alt="Equation 2" src="/files/ftp_upload/58076/58076eq2.jpg"> 참고:615,652및720 는 분 광 광도 계의 선형 흡 광도 범위에 적합 해야 한다. 필요한 경우, PBS 버퍼와 샘플을 희석.</li>
	</ol>
</li>
	<li>원래 샘플에 안료 농도 다시 계산. 샘플에서 안료 농도 방정식 (1) 및 (2) 사용 될 때 1 mL의 문화 및 추출 버퍼의 1 mL는 분석의 결과와 직접 해당 합니다. 박테리아 샘플 PBS 버퍼의 다른 볼륨을 사용 하는 경우 최종 안료 농도 식 (3)에 따라 계산 해야: <img alt="Equation 3" src="/files/ftp_upload/58076/58076eq3.jpg">(3) 참고: 셀 건조 중량 당 정규화 phycobiliprotein 콘텐츠, 경우 최종 안료 농도 계산 해야 방정식 (4)에 따라<img alt="Equation 4" src="/files/ftp_upload/58076/58076eq4.jpg"> </img> (4)</li>
</ol>5. (선택 사항) 셀 건조 중량의 결정
<ol><li>3 빈 안전 잠금 튜브 분석 균형에 무게. 주의: 그것은 중요 안전 잠금 튜브는 건조입니다. 젖은 환경에 튜브를 저장 하는 경우 동결 건조 하기 전에 그들을 무게에 몇 시간 동안 튜브를 건조. 부드럽게 튜브를 조작 및만 손으로 파우더 무료 장갑 낀 자료와 연구자의 손가락 사이 어떤 접촉을 피하기 위해. 모든 소유자를 유지 하 고 깨끗 한 관에 자료의 전송을 방지 하기 원심.</li>
	<li>1 x 15 mL 15 mL 원뿔 튜브에 문화 현 탁 액의 샘플.</li>
	<li>각 포함 15 mL의 물에 15 mL 원뿔 튜브 4000 x g 에서 10 분 원심 분리기로 터의 균형에 대 한 실험실 온도에서 세 개의 추가 15 mL 원뿔 튜브에에서 문화의 정지 원심 원심, 후는 상쾌한의 12 mL를 삭제 합니다.</li>
	<li>나머지 상쾌한에 펠 릿을 resuspend 하 고 혼합물의 3 x 1 mL를 피펫으로 3 1.5 mL 안전 잠금 튜브 전송. 펠 릿의 일부 남은 15 mL 원뿔 튜브에 남아, 경우에 이온된 수의 1.5 mL 원뿔 튜브, 소용돌이 또는 악수 튜브 나머지 resuspend를 작은, 및 혼합물의 3 x 0.5 mL 3 1.5 mL 안전 잠금 튜브 (이미 containi 전송에 추가 ng는 펠 릿의 3 x 1 mL)는 피 펫으로. 참고: 세 개의 1.5 mL 안전 잠금 튜브에 펠 릿을 나누어 건조 중량 측정의 기술적 오류 추정 수 있게 됩니다.</li>
	<li>원심 셀 5 분 균형에 대 한 실험실 온도에서 15000 x g 에서 1.5 mL 안전 잠금 튜브에 물 1 mL를 포함 하는 추가 1.5 mL 안전 잠금 튜브 원심 분리기 회전자. 원심, 후는 supernatants 삭제 합니다.</li>
	<li>냉동 실에 샘플을 넣어. 장기 저장을 위한-80 ° C에서 샘플을 계속 단기 저장용-20 ° C 충분 하다.</li>
	<li>Freeze-dry 샘플 하룻밤. 적절 한 동결 과정에 대 한 동결 건조 기 콘덴서-90 ° c의 온도 유지 하 고 동결 건조 기에서 압력 챔버 1 hPa.</li>
	<li>동결, 후 튜브를 닫고 샘플 분석 균형에 무게. 참고: 건조 중량 값 셀 건조 중량 당 phycobiliprotein 콘텐츠 정상화에 대 한 사용할 수 있습니다.</li>
</ol>

<ol>
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PCC 6803 autotrophic growth in a flat-panel photobioreactor.</a> Engineering in Life Sciences. 15 (1), 122-132 (2015).</li><li>Hemlata, G., Fareha, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+Anabaena+sp.+nccu-9+with+special+reference+to+phycocyanin.">Studies on Anabaena sp. nccu-9 with special reference to phycocyanin.</a> Journal of Algal Biomass Utilization. 2 (1), 30-51 (2011).</li><li>Rito-Palomares, M., Nuez, L., Amador, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+application+of+aqueous+two-phase+systems+for+the+development+of+a+prototype+process+for+c-phycocyanin+recovery+from+Spirulina+maxima.">Practical application of aqueous two-phase systems for the development of a prototype process for c-phycocyanin recovery from Spirulina maxima.</a> Journal of Chemical Technology &amp; Biotechnology. 76 (12), 1273-1280 (2001).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenium-Containing+Allophycocyanin+Purified+from+Selenium-Enriched+Spirulina+platensis+Attenuates+AAPH-Induced+Oxidative+Stress+in+Human+Erythrocytes+through+Inhibition+of+ROS+Generation.">Selenium-Containing Allophycocyanin Purified from Selenium-Enriched Spirulina platensis Attenuates AAPH-Induced Oxidative Stress in Human Erythrocytes through Inhibition of ROS Generation.</a> Journal of Agricultural and Food Chemistry. 59 (16), 8683-8690 (2011).</li><li>Nedbal, L., Trt&#237;lek, M., Cerven&#253;, J., Kom&#225;rek, O., Pakrasi, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photobioreactor+system+for+precision+cultivation+of+photoautotrophic+microorganisms+and+for+high-content+analysis+of+suspension+dynamics.">A photobioreactor system for precision cultivation of photoautotrophic microorganisms and for high-content analysis of suspension dynamics.</a> Biotechnology and Bioengineering. 100 (5), 902-910 (2008).</li><li>Zav&#345;el, T., Knoop, H., Steuer, R., Jones, P. R., &#268;erven&#253;, J., Trt&#237;lek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+evaluation+of+ethylene+production+in+the+recombinant+cyanobacterium+Synechocystis+sp.+PCC+6803+harboring+the+ethylene-forming+enzyme+by+membrane+inlet+mass+spectrometry.">A quantitative evaluation of ethylene production in the recombinant cyanobacterium Synechocystis sp. PCC 6803 harboring the ethylene-forming enzyme by membrane inlet mass spectrometry.</a> Bioresource Technology. 202, 142-151 (2016).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Lawrenz, E., Fedewa, E. J., Richardson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+protocols+for+the+quantification+of+phycobilins+in+aqueous+phytoplankton+extracts.">Extraction protocols for the quantification of phycobilins in aqueous phytoplankton extracts.</a> Journal of Applied Phycology. 23 (5), 865-871 (2011).</li><li>Lea-Smith, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phycobilisome-Deficient+Strains+of+Synechocystis+sp.+PCC+6803+Have+Reduced+Size+and+Require+Carbon-Limiting+Conditions+to+Exhibit+Enhanced+Productivity.">Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require Carbon-Limiting Conditions to Exhibit Enhanced Productivity.</a> Plant Physiology. 165 (2), 705-714 (2014).</li><li>Seo, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isolation+of+phycocyanin+from+Spirulina+platensis+associated+with+high-pressure+extraction+process.">Stable isolation of phycocyanin from Spirulina platensis associated with high-pressure extraction process.</a> International Journal of Molecular Sciences. 14 (1), 1778-1787 (2013).</li><li>Touloupakis, E., Cicchi, B., Torzillo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioenergetic+assessment+of+photosynthetic+growth+of+Synechocystis+sp.+PCC+6803+in+continuous+cultures.">A bioenergetic assessment of photosynthetic growth of Synechocystis sp. PCC 6803 in continuous cultures.</a> Biotechnology for Biofuels. 8 (1), 133 (2015).</li><li>Touloupakis, E., Cicchi, B., Benavides, A. M. S., Torzillo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high+pH+on+growth+of+Synechocystis+sp.+PCC+6803+cultures+and+their+contamination+by+golden+algae+(Poterioochromonas+sp.).">Effect of high pH on growth of Synechocystis sp. PCC 6803 cultures and their contamination by golden algae (Poterioochromonas sp.).</a> Applied Microbiology and Biotechnology. 100 (3), 1333-1341 (2016).</li><li>Ishii, A., Hihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+AbrB-Like+Transcriptional+Regulator,+Sll0822,+Is+Essential+for+the+Activation+of+Nitrogen-Regulated+Genes+in+Synechocystis+sp.+PCC+6803.">An AbrB-Like Transcriptional Regulator, Sll0822, Is Essential for the Activation of Nitrogen-Regulated Genes in Synechocystis sp. PCC 6803.</a> Plant Physiology. 148 (1), 660-670 (2008).</li></ol>

저자는 공개 없다.

이 프로토콜 모델 cyanobacterium Synechocystis에서 phycobiliprotein 콘텐츠의 정량화에 대 한 간단한 빠르고 재현할 수 방법을 설명합니다. 셀 균질, 단백질 추출과 phycocyanin와 allophycocyanin 정량화의 몇 가지 메서드를 비교 하 고 마지막 프로토콜의 모든 단일 절차의 최적의 단계 조합을 나타냅니다. 대표적인 데이터, phycobiliproteins의 콘텐츠는 빛의 강도 증가에서 Synechocystis 셀에 정량 했다. 비록 ...

fPhycobiliproteins는 간결한 박테리아 (Cyanophyta)에서 광 수확 안테나와 여러 진 핵 taxa (Glaucophyta, Rhodophyta의 주요 구성 요소를 나타내는 수용 성 색소 단백질 복합물 , 및 Cryptophyta)1. 그들은 phycobilisomes 라는 supramolecular 단지도 주로 발생 하 고 Cryptophyta, 어디는 phycobiliproteins 지역화는 제외한 stromal 측에 광합성 세포 막의 표면에 일반적으로 연결 되는 thylakoid 루멘2. 네 가지 유형의 phycobiliproteins 날짜까지 확인 되었습니다: 코어 allophycocyanin 및 주변 phycocyanin, phycoerythrin, 및 phycoerythrocyanin1. 주요 빛 수확 단지로 phycobilisomes 조류와 남조류의 대량 문화 생산성의 중요 한 요소 중 하나를 나타냅니다. 그것은 그 phycobilisomes 잘림 강한 빛3바이오 매스 축적을 향상 시킬 수 입증 되었습니다. 다른 한편으로, 겸손 또는 낮은 irradiance에서 안테나 잘림 결과 성장 속도 바이오 매스 축적 감소3,4. Phycobiliproteins는 상업적으로 사용 식용 염료, 의약품, 그리고 화장품 산업, 식품 첨가물, 형광 프로브 cytometry, 형광 immunoassays 및 형광 현미경 검사 법5에서 응용 프로그램과 함께.
이 프로토콜 phycobiliproteins 모델 cyanobacterium Synechocystis에서 양적 결정에 초점을 맞추고. 남조류는는 초기 oxygenic 광합성 슬에; 그들은 2.4 십억 년6이상에 대 한 지구의 생물권을 형성 되었습니다. 그들은 질소, 탄소, 산소, 및 기타 요소의 세계 생물 지구 화학적 순환에 중요 한 역할을 한다. 박테리아, 중 단 세포 변형 Synechocystis 이후 전체 게놈으로 첫 번째 cyanobacterium 독특한 위치를 얻은 시퀀싱7,8, 그것은 자연스럽 게 외 인 DNA9, 변환 및 그것은 안정적이 고 비교적 빠른 성장10,11을 수행합니다. Synechocystis, 핵심 안테나 구성에서에서 allophycocyanin, 완전 한 막 단백질 연관 이며 연결 된 phycocyanin thylakoid 막 주변에 위치 하 고 있습니다.
이 프로토콜 내에서 phycobiliprotein 추출 및 정량화에 대 한 여러 가지 방법은 비교 합니다. Synechocystis, 뿐만 아니라 다른 박테리아 변종, Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira 를 포함 하 여 최종 추출 절차 성공적으로 적용 했다 빨강 조류 Porphyridium cruentumsp., 그리고 Nostoc sp., 그것은 또한 성공적으로 적용 했다. 따라서,이 프로토콜에 개발 하는 방법 phycobiliprotein 추출에 대 한 보편적인 방법으로 여겨질 수 있다. 높은 phycobiliprotein 엽록소에 잔류물의 낮은 콘텐츠 함께 수익률 제공 여기 추출 절차를 설명 된 테스트 추출 방법의 일부 높은 총 단백질 수율 결과, 비록는 phycobiliprotein 압축을 풉니다. 엽록소의 한 콘텐츠를 줄이는 올바른 phycocyanin와 allophycocyanin spectrophotometric 정량화에 대 한 필수적 이었다.
Phycobiliprotein 흡수 스펙트럼 다양 한 조류와 박테리아 종12,13,14,15,,1617 크게 다를 수 있으며 심지어 중 단일 박테리아 속18의 여러 변종. 따라서, 특정 파장 및 흡수 계수 phycocyanin 및 Synechocystis 에 allophycocyanin에 사용 된 다른 계통에 일반적으로 적용 되지 않습니다. 또한, Synechocystis 는 phycoerythrin와 일부 다른 조류와 박테리아에서 찾을 수 있는 phycoerythrocyanin를 포함 하지 않습니다. Phycobiliproteins 긴장 Synechocystis이외에 결정을 위해 각 변형에 대 한 spectrophotometric 방정식을 개별적으로 평가 하는 것이 좋습니다.
프로토콜 포함 2 개의 더 단계 (하룻밤 셀룰러 펠 릿 및 1 시간 단백질 추출의 동결), 비록 phycobiliproteins 정량화에 대 한 총 노동 시간 보다 2 시간 이상 이다.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Synechocystis sp. PCC 6803</td>
			<td style="width:417px;">Institut Pasteur, Paris, France</td>
			<td style="width:137px;">6803</td>
			<td style="width:627px;">Cyanobacterium strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roti-CELL PBS</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td>9143.1</td>
			<td>Phosphate-Buffered Saline (PBS) solution, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf safe-lock tubes&#160;</td>
			<td style="width:417px;">Eppendorf, Hamburk, Germany</td>
			<td>30120086</td>
			<td>Safe-lock tubes 1.5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VWR 80-Place Storage System</td>
			<td style="width:417px;">VWR International, Radnor, Pennsylvania, USA</td>
			<td>30128-282</td>
			<td>Holder for safe-lock tubes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RAININ 100 &#181;l -1000 &#181;l&#160;</td>
			<td>Mettler-Toledo, Columbus, Ohio, USA</td>
			<td>17014382</td>
			<td>Pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GP-LTS-A-1000&#181;L-/F-768/8</td>
			<td>Mettler-Toledo, Columbus, Ohio, USA</td>
			<td>30389272</td>
			<td>Pipette tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotina 420R</td>
			<td>Hettich, Kirchlengern, Germany</td>
			<td>4701</td>
			<td>Refrigerated centrifuge for 1.5 ml safe-lock tubes and 15 ml conical centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LCexv 4010</td>
			<td>Liebherr, Bulle, Switzerland</td>
			<td>9005382197172</td>
			<td>Refrigerator and freezer -20&#160;&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Revco ExF -86&#176;C Upright Ultra-Low Temperature Freezer</td>
			<td style="width:417px;">Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td style="width:137px;">EXF24086V&#160;</td>
			<td>Freezer -80&#160;&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolSafe</td>
			<td>LaboGene, Liller&#248;d, Denmark</td>
			<td>7.001.000.615</td>
			<td>Freeze dryer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-2600</td>
			<td>Shimadzu, Kyoto, Japan</td>
			<td>UV-2600</td>
			<td>Spectrophotometer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hellma absorption cuvettes, semi Micro</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>Z600288&#160;</td>
			<td>VIS/UV-VIS semi-micro cuvettes 0.75-1.5 ml, spectral range 200-2500 nm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silamat S6</td>
			<td>Ivoclar Vivadent, Schaan, Liechtenstein</td>
			<td>602286WU</td>
			<td>Homogenizer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solid-glass beads</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>Z273627</td>
			<td>Glass bead of the diameter 2&#160;mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CPA225D-0CE</td>
			<td>Sartorius AG, G&#246;ttingen, Germany</td>
			<td>SECURA225D-1OBR</td>
			<td>Analytical balances</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C-Phycocyanin from Spirulina sp.&#160;</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>P2172</td>
			<td>Phycocyanin standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Allophycocyanin</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>A7472</td>
			<td>Allophycocyanin standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Bicinchoninic Acid Kit&#160;</td>
			<td style="width:417px;">Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td style="width:137px;">BCA1, B9643</td>
			<td>Complete kit for total proteins determination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">AlgaeTron&#160;</td>
			<td style="width:417px;">Photon System Instruments Ltd., Dr&#225;sov, Czech Republic</td>
			<td style="width:137px;">AG 130-ECO&#160;</td>
			<td>Cultivation chamber for E. flasks, with controllable light and atmosphere</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Photobioreactor</td>
			<td style="width:417px;">Photon System Instruments Ltd., Dr&#225;sov, Czech Republic</td>
			<td style="width:137px;">FMT-150</td>
			<td>Cultivation equipment for cyanobacteria and algae with completely controllable environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellometer&#160;</td>
			<td style="width:417px;">Nexcelom Bioscience, Lawrence, Massachusetts, USA</td>
			<td style="width:137px;">Auto M10</td>
			<td>Cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Corning 15 mL centrifuge tubes</td>
			<td style="width:417px;">Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td style="width:137px;">CLS430791&#160;</td>
			<td>15 ml Centrifuge tube for dry weigth sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herasafe KS</td>
			<td style="width:417px;">Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td style="width:137px;">51024579</td>
			<td>Laminar flow hood</td>
		</tr>
	</tbody>
</table>

spectrophotometric determination of phycobiliprotein content in cyanobacterium synechocystis

이것은 모델 cyanobacterium Synechocystisphycobiliprotein 콘텐츠의 양적 결정에 대 한 간단한 프로토콜 이다. Phycobiliproteins 있습니다 phycobilisomes, 박테리아에서 주요 광 수확 안테나의 가장 중요 한 구성 요소와 여러 조류 taxa. Synechocystis 의 phycobilisomes 포함 두 phycobiliproteins: phycocyanin와 allophycocyanin. 이 프로토콜은 간단 하 고, 효율적인, phycocyanin와이 모델 cyanobacterium에서 allophycocyanin의 양적 결정에 대 한 신뢰할 수 있는 방법을 설명합니다. 우리는 phycobiliprotein 추출 및 spectrophotometric 정량화의 몇 가지 메서드를 비교. 이 프로토콜에서 설명 된 대로 추출 절차는 Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., 같은 다른 박테리아 변종에도 성공적으로 적용 했다 그리고 Nostoc sp., 또한 레드 조류 Porphyridium cruentum로. 그러나, 다양 한 taxa에서 특정 phycobiliproteins의 소멸 계수 다를 수 있습니다 그리고 그것은, 따라서, 개별적으로 모든 단일 스트레인에 대 한 spectrophotometric 정량화 방법의 유효성을 검사 권장. 프로토콜은 시간이 좀 필요 하 고 그것만 표준 장비를 필요로 하기 때문에 모든 표준 생명 과학 실험실에서 수행할 수 있습니다.

초기 메서드 테스트에 대 한 Synechocystis BG11 재배 매체20 (17 mM HEPES 보충) 25 ° C, 50 µmol (광자)의 농도의 따뜻한 하얀 빛 아래에서 재배 통에 삼각 플라스 크에 배치 문화로는 / (m 2·s)와 1% CO2 자란 분위기에. 재배 기간 동안 문화 안전 잠금 튜브로 샘플링 된 centrifuged (15000 x g 5 분 동안 실험실 온도에서)는 상쾌한, 폐기 및 샘플 중...

Watch this Scientific Journal Video about Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis at JoVE.com

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

여기, 선물이 양적 phycobiliprotein cyanobacterium Synechocystis spectrophotometric 메서드를 사용 하 여에서 콘텐츠를 결정 하는 프로토콜. 추출 프로시저 또한 성공적으로 다른 박테리아 및 조류 긴장;에 적용 했다 그러나, 안료 흡수 스펙트럼에 있는 변이 때문 그것은 각 스트레인에 대 한 spectrophotometric 방정식을 개별적으로 테스트 하는 데 필요한입니다.

Spectrophotometric Cyanobacterium Synechocystis 에서 Phycobiliprotein 콘텐츠 결정

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

spectrophotometric-determination-phycobiliprotein-content

Research

JoVE Journal

Biochemistry

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

15.1K 조회수.  Czech Academy of Sciences. 여기, 선물이 양적 phycobiliprotein cyanobacterium Synechocystis spectrophotometric 메서드를 사용 하 여에서 콘텐츠를 결정 하는 프로토콜. 추출 프로시저 또한 성공적으로 다른 박테리아 및 조류 긴장;에 적용 했다 그러나, 안료 흡수 스펙트럼에 있는 변이 때문 그것은 각 스트레인에 대 한 spectrophotometric 방정식을 개별적으로 테스트 하는 데 필요한입니다.

비디오: Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

효능 판별 분석에서 펩타이드 작용제의 직렬 희석에 대한 분배 자동 음향

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

연구

생화학

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

시아 노 박테리아의 글리코겐 함량 측정

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

높은 처리량, 양적 도트를 사용 하 여 조직 수준에서 선택 된 단백질의 내용의 절대 결정 오 점 분석 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

식물에 철 분 함량을 측정 하는 색도계 방법

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

척도 부착 셀에 대상 참여를 이미징 높은 콘텐츠를 사용 하 여

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

마이크로 로르 나를 사용한 네이티브 단백 입자의 농축과 그에 따른 절대/상대 미세 유 변 함량 및 셀룰러 전송 속도의 후속 측정

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)&#8212;two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 &#177; 3.6 &#181;M and a Ki of 23.2 &#177; 3.2 &#181;M. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.

세포성 글루타티온 S-트랜스퍼라제의 잠재적 억제제에 대한 분광측정 검사

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.

단백질 지형 변화의 측정을 위한 단백질의 빠른 광화학 산화에 실시간 보상 가능하게

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

질량 광합성에 의한 항체 항원 선호도의 신속한 결정

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

다목적 미세 유체 칩의 직렬 결정학에 의한 효소의 결정화 및 구조적 결정: 기질 복합체

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...