Name: quantification of hypopigmentation activity in vitro
Uploaded: 2019-03-06T12:30:00
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이 작품은 한국 연구소의 계획과 기술 음식, 농업 및 임업 (IPET) 농업, 음식, 및 농촌 업무 (MAFRA 투자 농업-한국 기술 개발 프로그램을 통해에 대 한 평가 의해 지원 되었다 ) (116159-02-2-WT011) BK21 플러스, 고려 대학교 생명 공학 및 생명 과학의 학교.

1. 매체, 화합물, 및 시 약의 준비
<ol><li>B16F10 성장 전체 매체를 준비 합니다. 보충 Dulbecco의 10% 태아 둔감 한 혈 청 (FBS)와 1% 페니실린/스 (해충)이 글의 중간 (DMEM) 수정.<ol>
<li>완전 한 매체의 500 mL을 FBS와 1 패 멸 균된 유리병에 해충의 5 mL 50 mL와 Dulbecco의 수정된이 글의 중간 (DMEM)의 445 mL를 혼합 한다. 부드럽게 혼합 후 새로운 소독된 유리 병을 4 ° c.에 다음 저장소 0.2 µ m 병 상위 필터를 통해 매체 ...

<ol>
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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=Dual+inhibition+of+&#947;-oryzanol+on+cellular+melanogenesis:+inhibition+of+tyrosinase+activity+and+reduction+of+melanogenic+gene+expression+by+a+protein+kinase+a-dependent+mechanism.">Dual inhibition of &#947;-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase a-dependent mechanism.</a> Journal of Natural Products. 75 (10), 1706-1711 (2012).</li><li>Choi, Y. M., 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=Effects+of+the+isoflavone+puerarin+and+its+glycosides+on+melanogenesis+in+B16+melanocytes.">Effects of the isoflavone puerarin and its glycosides on melanogenesis in B16 melanocytes.</a> European Food Research and Technology. 231 (1), 75-83 (2010).</li><li>Cho, B. R., Jun, H. J., Thach, T. T., Wu, C., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaine+reduces+cellular+melanin+content+via+suppression+of+microphthalmia-associated+transcription+factor+in+B16-F1+murine+melanocytes.">Betaine reduces cellular melanin content via suppression of microphthalmia-associated transcription factor in B16-F1 murine melanocytes.</a> Food Science and Biotechnology. 26 (5), 1391-1397 (2017).</li><li>Overwijk, W. W., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B16+as+a+mouse+model+for+human+melanoma.">B16 as a mouse model for human melanoma.</a> Current Protocols in Immunology. , (2001).</li><li>Virador, V. M., Kobayashi, N., Matsunaga, J., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+protocol+for+assessing+regulators+of+pigmentation.">A standardized protocol for assessing regulators of pigmentation.</a> Analytical Biochemistry. 270 (2), 207-219 (1999).</li><li>D'Mello, S. A. N., Finlay, G. J., Baguley, B. C., Askarian-Amiri, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+melanogenesis.">Signaling pathways in melanogenesis.</a> International Journal of Molecular Sciences. 17 (7), 1144 (2016).</li><li>Hou, L., Panthier, J. J., Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+and+transcriptional+regulation+in+the+neural+crest-derived+melanocyte+lineage:+interactions+between+KIT+and+MITF.">Signaling and transcriptional regulation in the neural crest-derived melanocyte lineage: interactions between KIT and MITF.</a> Development. 127 (24), 5379-5389 (2000).</li><li>Hedley, S. J., Gawkrodger, D. J., Weetman, A. P., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-MSH+and+melanogenesis+in+normal+human+adult+melanocytes.">&#945;-MSH and melanogenesis in normal human adult melanocytes.</a> Pigment Cell Research. 11 (1), 45-56 (1998).</li><li>Hu, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+evaluation+of+melanin+content+and+production+of+pigment+cells+in+vitro.">Methodology for evaluation of melanin content and production of pigment cells in vitro.</a> Photochemistry and Photobiology. 84 (3), 645-649 (2008).</li><li>Carriel, V. 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우리는 교양된 melanocytes을 사용 하 여 테스트의 hypopigmentation 활동을 평가 하기 위한 프로토콜을 제시. 대표 결과 알 부 틴, 저해 tyrosinase 활동 및 셀룰러 멜 라 닌 콘텐츠 tyrosinase 억제 물의 hypopigmentation 효과를 보여주었다. 이러한 메서드는 안티 melanogenic 활동 연구에 널리 사용 됩니다. 이 분석 실험을 사용 하 여, 또한 성공적으로 확인 했습니다 melanogenesis B16F10 세포에 억제 효과 과거에는 여러 가?...

멜 라 닌은 생리학, 병리학, 피부, 눈, 뇌1등 여러 장기의 독성에 중요 한 역할을 한다. 멜 라 닌의 주요 기능 사진 심사 및 생 화 확 적인 효과가 있습니다. 멜 라 닌 근처-적외선과 보이는 빛으로 자외선 (UV) 방사선, 짧은 파장의 빛;에서 증가 흡수 속도와 흡수 따라서, 멜 라 닌 가시광선 이나 자외선 방사선2로 인 한 손상 으로부터 조직을 보호 합니다. 멜 라 닌은 항 산화 및 금속과 다른 독성 화학 물질;에 대 한 선호도 따라서, 그것은 산화 화학 스트레스3에서 조직을 보호할 수 있습니다. 그러나, 멜 라 닌의 과도 한 생산 피부 문제를 발생합니다.
피부와 홍 채에 있는 melanin의 품질과 수량은 아이리스와 피부 색깔의 가장 중요 한 결정 요인. 개인은 다른 피부 색깔 기본 설정; 있을 수 있습니다. 다른 더 가벼운 피부 색깔을 선호 하는 동안 일부 검게 그을린된 피부를 선호 합니다. 이러한 소비자 프로 파일에 따라 hypopigmentation 화장품 선호 피부 색상4에 대 한 개별 시장을 만족 시키기 위해 개발 되었습니다. 따라서, hypopigmentation 및 안티 melanogenic 활동의 연구 모두 과학적으로 그리고 실제적으로 중요 하다.
Melanogenesis는 멜 라 닌 생 합성 효소 및 자발적인 화학 반응을 melanocytes에 일련의 복잡 한 프로세스입니다. 한 melanocyte 약 36 keratinocytes 둘러싸여 있으며 melanocytes 이웃 keratinocytes 그들의 제품을 배포 하는 멜 라 닌 합성 공장. 피부에 멜 라 닌을 생산 하 고 melanocytes의 melanosomal 구획에 저장 dendrites 통해 표 피에에서 인접 keratinocytes에 수송 된다.
L-티로신 melanogenesis 및 효소 tyrosinase 초기 기판 catalyzes L-티로신 그리고 dopaquinone로 3, 4-dihydroxyphenylalanine (DOPA)으로 변환 하는 두 개의 연속 반응을 제공 합니다. 이러한 반응 속도 제한 단계 melanogenesis5,6에 있습니다. 따라서, hypopigmentation 활동 세포 tyrosinase 활동을 직접 평가 하 여 먼저 측정할 수 있습니다. 이렇게 하려면 melanocyte 추출 tyrosinase를 포함 하는 인 큐베이 팅 DOPA와 및 dopaquinone 샘플에서 475에 분 광 광도 법으로 측정 될 수 있다 nm. 값은 샘플, 및 컨트롤에 비해 적은 dopaquinone 형성에 hypopigmentation 활동 결과와 물질의 단백질 농도 의해 정규화 됩니다.
둘째, hypopigmentation 활동 교양된 melanocytes에 멜 라 닌을 직접 측정 하 여 측정할 수 있습니다. 시험 체와 세포 치료, 후 멜 라 닌 알칼리 조건 하에서 추출 되 고 멜 라 닌 콘텐츠 400 분 광 광도 법으로 정량 nm. Hypopigmentation 에이전트 컨트롤7보다 더 낮은 melanin 내용을 발생 합니다.
마지막으로, hypopigmentation 활동 폰 타나-마 송 melanin 얼룩 및 후속 이미지 분석 하 여 측정할 수 있습니다. 폰 타나-마 송 얼룩에 멜 라 닌과 립을 보이는 검은 금속 상태로 암모니아-실버 질산염을 줄일 하 고 미세한 이미지 셀의 검정 영역은 멜 라 닌의 양을 나타내며.
Hypopigmentation 에이전트 보통 제공 일관 되 고 비교 결과이 세 가지 방법으로는 물질의 활동 올바른지 확인 합니다. 또는, 그것은 주요 유전자와 단백질 melanogenesis hypopigmentation 활동을 조사 하는 시험 물질에 대 한 응답에서에 식을 측정 하기 유용할 수 있습니다. Tyrosinase, 뿐만 아니라 tyrosinase 관련 (TRP-1) 단백질과 dopachrome tautomerase (TRP-2) melanogenesis8에 중요 한 효소는. 녹음 방송 요인 microphthalmia 관련 된 전사 요소 (MITF) melanogenesis는 유전자/단백질 식 레벨의 정량화에 마스터 레 귤 레이 터 또는 발기인 활동 분석 결과 hypopigmentation 활동을 평가 하기 위해 사용할 수 있습니다.

<table>
	<tbody>
		<tr>
			<td>3,4-Dihydroxy-l-phenylalanine</td>
			<td>Sigma</td>
			<td>D9628</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Sigma</td>
			<td>#320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Arbutin</td>
			<td>Fluka</td>
			<td>#10960</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Yakuri Pure Chemicals</td>
			<td>#16223</td>
			<td>37%</td>
		</tr>
		<tr>
			<td>Gold chloride</td>
			<td>American MasterTech</td>
			<td>AHG0226</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Samchun chemical</td>
			<td>H0255</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bio basic Canada Inc.</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>#60218</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>J.T.Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duksan pure chemicals</td>
			<td>#81</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>655104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear fast red</td>
			<td>Merck</td>
			<td>100121</td>
			<td>Nuclear fast red 0.1% in 5% aluminum sulfate.</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>J.T. Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate pentahydrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#2163</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Bio Basic Canada</td>
			<td>TB0196</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Union Carbide</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosinase from mushroom</td>
			<td>Sigma</td>
			<td>T3824</td>
			<td>25 KU</td>
		</tr>
	</tbody>
</table>

quantification of hypopigmentation activity in vitro

이 연구는 생체 외에서 hypopigmentation 활동의 정량화에 대 한 실험실 방법을 선물 한다. Melanocytes에 주요 안료 멜 라 닌은 여러 세포와 환경 요인에 대 한 응답에서 합성 됩니다. 멜 라 닌 자외선 손상 으로부터 피부 세포를 보호 하지만, 또한 생물 및 생 화 확 적인 기능을가지고. 과도 한 생산 또는 축적 melanocytes에 멜 라 닌의 주 근 깨, 검 버섯, melasma, 사마귀 등 피부 문제를 일으킬 수 있습니다. 따라서, hypopigmentation 에이전트 melanogenesis 제어 임상 또는 화장품 요구와 개인에서 중요 하다. 멜 라 닌은 주로 외부 영향 melanogenesis 라는 복잡 한 생 화 확 적인 과정에 내재 성 요인, 호르몬, 염증, 나이, 등 자외선 빛 노출 melanocytes melanosomes에 합성 됩니다. 우리는 화학 물질 또는 melanocytes에 자연 물질의 hypopigmentation 활동을 결정 하는 세 가지 방법을 설명: 1) 셀룰러 tyrosinase 활동 및 2) 멜 라 닌 콘텐츠 및 이미지 3) 얼룩과 측량 셀룰러 멜 라 닌의 측정 분석입니다.
Melanogenesis에서 tyrosinase L-티로신 그리고 dopaquinone로 3, 4-dihydroxyphenylalanine (l 아미노산의 일종)으로 변환 하는 속도 제한 단계 catalyzes. 따라서 tyrosinase의 저해는 기본 hypopigmentation 메커니즘입니다. 교양된 melanocytes에 tyrosinase 활동 기판으로 아미노산의 일종을 추가 하 고 dopaquinone 생산 분 광 광도 법으로 측정 하 여 측정할 수 있습니다. Melanogenesis 또한 멜 라 닌 콘텐츠를 측정 하 여 측정할 수 있습니다. 포함 하는 멜 라 닌 세포 일부는 NaOH와 함께 추출 되 고 멜 라 닌 spectrophotometrically 계량. 마지막으로, 멜 라 닌 콘텐츠 폰 타나-마 송 멜 라 닌의 얼룩이 다음 이미지 분석에 의해 측정할 수 있습니다. 이러한 생체 외 분석 실험의 결과 항상 복제 될 수 없다 인간의 피부에, 비록 이러한 메서드는 널리 특히 잠재적인 hypopigmentation 활동을 식별 하는 초기 단계로 melanogenesis 연구에 사용. 이 방법 또한 melanocyte 활동, 성장과 분화를 평가 하기 위해 사용할 수 있습니다. 세 가지 다른 방법으로 일관 된 결과 효과의 유효성을 확인합니다.

알 부 틴, 안티-melanogenic의 hypopigmentation 활동에 대 한 대표적인 결과 화합물, B16F10 melanocytes 다음과 같습니다. 그림 1A 는 그 알 부 틴은 크게 차량 처리 제어에 비해 세포 tyrosinase 활동 억제를 보여 줍니다. 마찬가지로, 알 부 틴과 자극된 세포의 멜 라 닌 콘텐츠 컨트롤 (그림 1B)에 비해 감소 크게 되었다. 셀 폰 타나-마 송 얼룩과 ?...

Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Quantification of Hypopigmentation Activity In Vitro

체 외 화학 물질의 hypopigmentation 활동을 평가 하기 위한 세 가지 실험 방법 설명: 1) 셀룰러 tyrosinase 활동과 2) 멜 라 닌 콘텐츠, 및 3) 멜 라 닌 세포의 멜 라 닌 얼룩 및 이미지 분석에 의해 측정의 정량화.

생체 외에서 Hypopigmentation 활동의 정량화

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

이 방법은 항 멜라노겐성 활동을 가진 기능성 화장품 및 피부제의 개발에 도움이 될 수 있다. 그것은 수행해야하고 결과를 신속하게 얻을 수있다. 또한, 이 방법은 연구자에게 유용할 수 있으며, 이들은 효과 나 화합물을 식별하거나 조사하거나 antimelanogenic 또는 프로멜라노겐성 활동을 조사하려는 사람들에게 유용할 수 있다.티로시나아제 활성을 측정하기 위해 B16-F10 멜라니시드제를 세포 배양 인큐베이터에서 24시간 동안 완전한 배지에서 24웰 플레이트 농도의 웰당 5배 10~4번째 세포로 시드하는 것으로 시작한다. 다음 날, 갓 준비된 테스트 화합물 및 억제제 또는 음의 제어로 보충된 DMEM으로 각 우물의 상체를 교체하고 플레이트를 인큐베이터로 72시간 동안 반납한다. 치료가 끝나면 우물을 두 번 헹구고 300 마이크로리터의 차가운 PBs를 잘 헹구고 접시를 얼음 위에 놓습니다.다음으로 셀 스크레이퍼를 사용하여 각 웰에 300 마이크로리터의 용해 버퍼를 추가하여 5분 후에 셀 용액을 수집합니다. 결과 세포 입자 현탁액을 개별 1.5 밀리리터 미세 원심분리기 튜브로 전달합니다. 그리고 세포 간 티로시나아제를 방출하기 위해 얼음에 14, 500 rpm에서 2 초 동안 3 번 용해.원심분리에 의해 세포 파편을 펠렛하고 얼음에 개별 1.5 밀리리터 원심분리기 튜브에 슈퍼 네이션을 전송합니다. 명확한 96 개의 잘 폴리스티렌 마이크로 플레이트의 개별 우물에 각 수퍼티의 70 마이크로 리터를 할당하고 140 개의 미세 리터의 티로시나제 기판 용액을 각 수퍼 나탄에 추가합니다. 접시를 부드럽게 흔들어 잘 내용을 혼합합니다.그런 다음 접시를 섭씨 37도에서 2시간 동안 배양합니다. 그리고 마이크로 플레이트 판독기에서 475 나노미터에서 티로시나제 활성을 측정합니다. 치료된 세포의 리세이트를 수집한 후 세포의 멜라닌 함량을 측정하기 위해 입증하였다.원심분리로 용액을 수집하고 수퍼네이션을 새로운 튜브로 옮긴다. 각 펠릿에 일반 수산화 나트륨 300마이크로리터를 섭씨 60도에서 1시간 배양합니다. 이어서 용존 셀 현탁액의 원심분리.그런 다음 슈퍼나티의 200 마이크로리터를 96웰 플레이트의 각 웰에 옮기합니다. 그리고 400 나노미터 파장에서 마이크로 플레이트 판독기의 멜라닌 함량을 측정합니다. 폰타나-마슨 스테닝의 경우, 300 마이크로리터의 콜드 PBs로 처리된 세포를 두 번 세척하고 섭씨 4도에서 1시간 동안 10%의 미공식적으로 200마이크로리터로 세포를 고정합니다.증류수로 두꺼운 세포를 헹구고 58~68도의 마이크로리터 200개를 각 우물에 암모니아칼 실버 작업 용액을 추가합니다. 섭씨 37도에서 1시간 동안 배양하거나 세포가 황갈색이 될 때까지. 증류수로 얼룩진 세포를 헹구고 실온에서 0.1%의 금염화물로 2분 배양합니다.금 염화물 처리 된 세포를 증류수로 헹구고 세포에 200 마이크로 리터의 나트륨 티오툴페이트 용액을 추가하십시오. 2 분 후에 세포를 다시 헹구십시오. 그리고 5 분 동안 잘 당 핵 고속 적색의 200 마이크로 리터로 세포를 레이블.그런 다음 빛 현미경으로 이미징하기 전에 수돗물로 얼룩진 세포를 씻으릅니다. 임계값 분석의 경우 이미지 J에서 이미지를 열고 이미지를 선택하고 이미지, 조정 및 색상 임계값을 선택합니다. 폰타나 매슨으로 염색된 영역을 측정하기 위해 밝기 매개변수 막대를 0으로 설정하고 모든 검은색 또는 갈색 스테인드 셀이 포함된 지점으로 밝기를 조정합니다.파티클 분석 및 분석을 선택하고 파티클 분석 창에서 요약 상자를 확인한 다음 확인을 클릭하여 결과의 요약을 얻고 데이터를 스프레드시트에 복사하고 붙여넣습니다. Arbutin 처리는 대조군 차량 처리 세포에 비해 세포 티로시나제 활성을 현저하게 억제합니다. 마찬가지로 Arbutin으로 자극된 세포의 멜라닌 함량은 대조군에 비해 현저히 감소된다.세포는 치료 단 사이에서 형태학적으로 유사하더라도, Arbutin는 대조군 처리된 세포에 비해 검은 색소의 면적을 감소시다. 이 방법은 복합 라이브러리를 사용하여 스크리닝하는 데 유용합니다. 신속하게 테스트할 샘플의 수백 이상이 있습니다.또한이 방법은 멜라노발생과 관련된 메커니즘을 조사하는 데사용할 수 있습니다. 생리 활성 후보 화합물이 확인 된 후, 생물학적 메커니즘 또는 상업적 응용 프로그램의 연구에 대한 자신의 증언.

quantification-of-hypopigmentation-activity-in-vitro

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

의 pH에 ​​의존 활동의 검출<em&gt; 대장균</em&gt; 보호자 HdeB<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

연구

생화학

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

풍부 하 고 대장균 에서 전송 RNAs의 레벨 충전의 정량화

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

매핑 대사: 조직에서 직접 젖 산 효소 활동 모니터링

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

TRNA Isopentenyl 전이 효소 활동을 감지 하는 체 외에서 분석 결과

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

생체 외에서 교정 무료 단백질 호모-oligomerization 상업 계측 및 무료, 오픈 소스 밝기 분석 소프트웨어를 사용 하 여의 정량화

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

초파리 멜라노가스터의 구연산 신타제 활동의 대색 분석

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

케노하브디티스 예쁜꼬마선충에서 2-아라키도노일글리세롤의 정량화를 위한 신분화 표준의 합성

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

멀티플렉스 동시 면역 침전을 이용한 단백질 상호 작용 네트워크 역학의 정량화

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

세포와 조직에서 코엔자임 A의 정량화

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

mtDNA의 합성 및 분포에 대한 고처리량 정량화

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

저자 스포트라이트: 미토콘드리아 DNA 합성 및 분포의 고처리량 이미지 기반 정량화  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

미토콘드리아 DNA 합성 및 분포의 고처리량 이미지 기반 정량화

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...