Name: enrichment of detergent-insoluble protein aggregates from human postmortem brain
Uploaded: 2017-10-24T12:30:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

저자는 박사 짐 Lah와 앨런 Levey, 모리 부 신경과의 도움이 의견 및 제안에 대 한 감사합니다. 이 작품은 부분적으로 투자 함으로써 의학 협력 가속 부여 (U01AG046161-02)에 모리 Alzheimer의 질병 연구 센터 (P50AG025688) 및 노후화에 국가 학회 부여 (R01AG053960-01) N.T.S.를 이 연구도 일부 모리 신경 과학 NINDS 핵심 시설 보조금, P30NS055077의 Neuropathology 코어에 의해 지원 되었다.

윤리 성명: 모든 뇌 조직에 모리 츠에서 가져온 &#39; s 질환 연구 센터 (ADRC) 두뇌 은행. 인간의 사후 조직 적절 한 제도적 검토 위원회 (IRB) 프로토콜에서 인수 했다.
 1. 균질 및 분류 <ol><li>조직 선택 참고: 사후 담당 조직에서 건강 한 제어 (Ctl) 및 병 적 고정 광고 사례를 확인된은 모리에서 선정 되었다 ADRC 두뇌 은행 (n = 2). 녹말 체 패 배포의 사후 neuropathological 평가 츠에 대 한 레지스트리를 설정 하는 컨소시엄에 따르면 수행 했다 &#39; s 질병 반 정량적 기준 24, 비록 득점 neurofibrillary 얽힌 병리학 Braak 준비 시스템에 따라 평가 했다. 16 <ol><li>얻기 냉동 사후 뇌 조직에서 건강 한 제어 (Ctl) 및 병 적 확인 광고의 경우. 드라이 아이스, 집게 및 면도날의 컨테이너를 활용 하 여, 소비 세 ~ 250mg 부분 tared 일회용에 각 조직 샘플에서 회색 물질의 무게 배, 조직 해빙 하지 않도록 돌보는. 무 균 수를 각 작품의 무게를 기록.</li>
		<li>소비 세 ~ 250mg의 회색 문제를 사용 하 여 집게와 면도기 시각적으로 검사 하 여 외부 회색 물질과 내부 백색 질 사이의 인터페이스를 찾습니다. 백색 질 및 더 중요 한 것은, 어떤 큰 혈관 또는 살 벌 한 지역, meninges, 또는 거미 집 모양의 메이 터. 신속 하 게 작업 하 고 자주 폴리스 티 렌 용기 마른 얼음에 뇌 조직으로 무게 배를 배치 하 여 절단 과정에서 냉동 조직 유지</li>
		<li>Excised 분석 균형을 사용 하 여 각 냉동된 조각에서 회색 물질의 정확한 무게를 기록 합니다. 계산 볼륨 낮은 소금 (LS) 버퍼의 dounce 균질 (5 mL/g 또는 20 %w / v)에 대 한 필요 (표 1 참조).</li>
		<li>프로 테아 제와 인산 가수분해 효소 억제 물 칵테일 및 얼음에 진정 x 1 준비 10 mL LS 버퍼</li>
		<li>무게 조직과 무게 보트에서 제거 드라이 아이스와 약 2 x 2 m m 조각으로 주사위로 그것은 녹은. 얼음에 2 mL 미리 냉장된 dounce 균질 화기 튜브에 전송</li>
		<li>프로 테아 제와 인산 가수분해 효소 억제 물 칵테일 X 1와 차가운 낮은 소금 (LS) 버퍼의 5 mL/g (20 %w / v) 추가.</li>
		<li>Homogenize 높은 클리어런스 유 봉 A와 낮은 클리어런스 유 봉 b.의 15 획의 약 10 타를 사용 하 여 얼음에 뇌 조직</li>
		<li>유리 파스퇴르 피 펫을 사용 하 여 2 mL 폴 리 프로필 렌 튜브에 모든 homogenate를 전송. 와 함께 튜브 라벨 &#8220; 일 &#8221; (총 Homogenate), 조직의 경우 수, 그리고 homogenate 볼륨. 약 수 0.8 mL 레이블이 1.5 mL 튜브에. 어떤 과잉 homogenate 수 마찬가지로 aliquoted-80에 저장 ° c.</li>
		<li>각 0.8 mL aliquot, 추가 100 µ L 각 5 M NaCl 및 10% (w/v) sarkosyl의 농도 0.5 M 및 1 %w / v, 각각. 반전에 의해 잘 튜브를 혼합 하 고 15 분 레이블 튜브에 대 한 얼음에 품 어 &#34; TH-S &#34; (총 Homogenate-Sarkosyl)는 homogenates sarkosyl-버퍼 (표 1)에 나타냅니다.</li>
		<li>얼음에 30% 진폭에서 3 5의 펄스에 대 한 각 튜브를 sonicate (최대 강도 = 40%) 사용 하 여 microtip 프로브.</li> <li>bicinchoninic 산 (BCA)를 사용 하 여 homogenates의 단백질 농도 시험 25 확인 방법. 
		참고: 조직의 그램 당 5 mL LS에서 일 S homogenates의 평균 단백질 농도 15 ~ 20 mg/mL. homogenates 수 수 즉시 분류 한 또는 사용까지-80 ° C에 저장.</li>
		<li>10 mg/ml 1 프로 테아 제와 인산 가수분해 효소 억제제 x 차가운 사크 버퍼 (표 1)를 사용 하 여 희석 TH-S homogenates.</li>
		<li>전송 5 mg 단백질 (0.5 mL) 500 µ L 폴 리 카보 네이트 ultracentrifuge 튜브와 쌍-균형 사크 버퍼에 각 TH-S homogenate의. 미리 냉장된 회전자와 180000 x g 1.5 mL 튜브에 4 ° c. sarkosyl-수용 성 전송 supernatants (S1)에서 30 분에서 ultracentrifuge에 튜브를 로드 하 고-80에서 저장 ° c. 
		참고: 사크-버퍼는 ultracentrifuge의 (선택적 세척) 추가 200 µ L 튜브 세제 불용 성 분수 (P1)를 포함 하 고 200 µ L 피 펫 팁을 사용 하 여 ultracentrifuge 튜브의 아래쪽에서 펠 릿 (P1)를 꺼내 려.</li>
		2-3 s <li>짧게 펄스-스핀은 거꾸로 튜브 (&#8804; 2500 x g) 아래 1.5 mL 튜브에 펠 릿 (P1) 및 버퍼 전송 microcentrifuge에. 펠 릿 중단 되도록 아래로 pipetting으로 사크 버퍼에서 불용 성 펠 릿 (P1) resuspend.</li>
		<li>쌍-균형, 그리고 4에서 추가 30 분 180000 x g에서 원심 분리기 ° c.</li>
		<li>선택적 세척 상쾌한 (S2)를 폐기 하 고 1 요소 버퍼 (표 1)의 50-75 µ L에서 sarkosyl-불용 성 펠 릿 (P2)를 품 어 그림 (프로 테아 제와 인산 가수분해 효소 억제 물 칵테일) 실 온에서 30 분 동안 solubilize x는 펠 릿. 
		참고: 따뜻한 요소 버퍼 SDS 강수량을 피하기 위해 사용 하기 전에 실내 온도에. 세제에 민감한 애플리케이션을 위해 요소 버퍼에서 SDS를 생략 하 고 요소 버퍼에 resuspending 전에 낮은 소금 버퍼에 불용 성 펠 렛 (P2) 세척을 고려.</li>
		<li>0.5 mL 튜브에 resuspended 펠 릿 (P2)을 전송 하 고 간단한 사용 (1 s) 20% 진폭 microtip 쥡니다 (최대 강도 = 40%)을 완벽 하 게 알 약을 solubilize.</li>
		<li>Sarkosyl-수용 성 (S1)의 단백질 농도 결정 하 고-BCA를 사용 하 여 불용 성 (P2) 분수 시험 방법. 이 분수를 사용 하 여 즉시 또는 사용까지-80 ° C에 저장.</li>
	</ol></li></ol> 2. Immunoblotting <ol>서양 blotting <li> 참고: 약간의 수정을 이전에 보고 된 절차에 따라 수행 했다 서쪽 blotting. 5 , 10 <ol><li>준비 40 µ g 샘플 총 homogenates (TH-S), 사크 성 (S1) 및 5 mM의 TCEP ph 7 1 X Laemmli SDS 페이지 샘플 버퍼에 사크-불용 성 (P2) 분수의. 5 분 동안 95 ° C에서 샘플을 품 어</li>
		<li>부하 샘플에는 캐스트 10 잘 4-12% 두번째-트리 스 SDS 페이지 젤. 첫 번째 센티미터 (10 분)에 대 한 V 80와 120 V 나머지 (60 분)에 대 한 또는 추적 염료는 젤의 맨 아래에 도달할 때까지 electrophorese.</li>
		<li>신선한 면도날과 젤 칼 분할-오픈 프리 카스트 젤 카세트를 사용 합니다. 부드럽게 버려야 빗과 신선한 면도날으로 젤의 끝.</li>
		<li>Pipetting으로 그림 고 아래로 1 x 200 µ l 사크-버퍼에 불용 성 펠 릿 (P1) resuspend.</li>
		<li>0.2 µ m 니트로 막 더 럽 히 기계에 건조 전송 메서드를 사용 하 여 전송 (자료 표 참조).</li>
		<li>차단 BB와 뒤를 0.05% 블로킹 버퍼 (BB) 트윈 20 30 분 없이 막 30 분 트윈 20 (BB/T). 차단, 후 린스 TBS/T에서 막 (0.1% Tween 20) 5 분 초과 차단 버퍼 제거.</li>
		<li>준비 1:1,000 희석 주 항 체 pT231, 타우-2 (팬 타우), AT8의 그리고 BB/T에서 EEA1 (0.05% 트윈 20).</li>
		<li>원형 동요와 4 ° C에서 하룻밤 1 차적인 항 체 솔루션에 막 품 어. 3 x 15 분에 대 한 큰 술/T에 막 씻어</li>
		<li>어둠 통에 붙일 레이블 근처-적외선 이차 항 체의 BB/T에 실 온에서 60 분에 대 한 1:20,000 희석와 막 조사. Tbs.에 3 x 10 분 TBS/T와 2 x 5 분 막 씻어</li>
		<li>스캔 이미징 시스템 (예: 700 nm (빨강 채널) 적외선 염료 680 염소 반대로 마우스 IgG (H + L) 보조와 800 nm (녹색 채널) 적외선 염료 790 당나귀-토끼 IgG (H + L에 대 한 적절 한 여기 파장에는 적외선을 사용 하 여 막 보조)).</li>
		<li>이미징 소프트웨어를 사용 하 여 신호 강도 계량 제조자에 의하여 densitometry 측정 수행을 &#39; s 지시, 평균 전체 배경 설정 자동 배경 설정 보장.</li>
	</ol>
	</li>
</ol>

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	<li>Taylor, J. P., Hardy, J., Fischbeck, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+Proteins+in+Neurodegenerative+Disease.">Toxic Proteins in Neurodegenerative Disease.</a> Science. 296 (5575), 1991 (1991).</li><li>Ross, C. A., Poirier, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+aggregation+and+neurodegenerative+disease.">Protein aggregation and neurodegenerative disease.</a> Nature Medicine. 10, S10-S17 (2004).</li><li>Ram&#237;rez-Alvarado, M., Merkel, J. S., Regan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+exploration+of+the+influence+of+the+protein+stability+on+amyloid+fibril+formation+in+vitro.">A systematic exploration of the influence of the protein stability on amyloid fibril formation in vitro.</a> Proc. Natl. Acad. Sci. 97 (16), 8979-8984 (2000).</li><li>Hamley, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Amyloid+Beta+Peptide:+A+Chemist's+Perspective.+Role+in+Alzheimer's+and+Fibrillization.">The Amyloid Beta Peptide: A Chemist's Perspective. Role in Alzheimer's and Fibrillization.</a> Chem Rev. 112 (10), 5147-5192 (2012).</li><li>Diner, I., 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=Aggregation+Properties+of+the+Small+Nuclear+Ribonucleoprotein+U1-70K+in+Alzheimer+Disease.">Aggregation Properties of the Small Nuclear Ribonucleoprotein U1-70K in Alzheimer Disease.</a> J. Biol. Chem. 289 (51), 35296-35313 (2014).</li><li>Gozal, 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=Proteomics+Analysis+Reveals+Novel+Components+in+the+Detergent-Insoluble+Subproteome+in+Alzheimer's+Disease.">Proteomics Analysis Reveals Novel Components in the Detergent-Insoluble Subproteome in Alzheimer's Disease.</a> J. Proteome Res. 8 (11), 5069-5079 (2009).</li><li>Hales, C. 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=Changes+in+the+detergent-insoluble+brain+proteome+linked+to+amyloid+and+tau+in+Alzheimer's+Disease+progression.">Changes in the detergent-insoluble brain proteome linked to amyloid and tau in Alzheimer's Disease progression.</a> PROTEOMICS. , (2016).</li><li>Julien, C., Bretteville, A., Planel, E., Sigurdsson, E. M., Calero, M., Gasset, 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> Amyloid Proteins: Methods and Protocols. , 473-491 (2012).</li><li>Nizhnikov, A. A., 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=Proteomic+Screening+for+Amyloid+Proteins.">Proteomic Screening for Amyloid Proteins.</a> PLoS ONE. 9 (12), e116003 (2014).</li><li>Seyfried, N. T., 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=Quantitative+analysis+of+the+detergent-insoluble+brain+proteome+in+frontotemporal+lobar+degeneration+using+SILAC+internal+standards.">Quantitative analysis of the detergent-insoluble brain proteome in frontotemporal lobar degeneration using SILAC internal standards.</a> J. Proteome Res. 11 (5), 2721-2738 (2012).</li><li>Woltjer, R. L., 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=Proteomic+determination+of+widespread+detergent+insolubility,+including+A&#946;+but+not+tau,+early+in+the+pathogenesis+of+Alzheimer's+disease.">Proteomic determination of widespread detergent insolubility, including A&#946; but not tau, early in the pathogenesis of Alzheimer's disease.</a> FASEB J. , (2005).</li><li>Miake, H., Mizusawa, H., Iwatsubo, T., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+Characterization+of+the+Core+Structure+of+&#945;-Synuclein+Filaments.">Biochemical Characterization of the Core Structure of &#945;-Synuclein Filaments.</a> J. Biol. Chem. 277 (21), 19213-19219 (2002).</li><li>Hasegawa, 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=Phosphorylated+&#945;-Synuclein+Is+Ubiquitinated+in+&#945;-Synucleinopathy+Lesions.">Phosphorylated &#945;-Synuclein Is Ubiquitinated in &#945;-Synucleinopathy Lesions.</a> J. Biol. Chem. 277 (50), 49071-49076 (2002).</li><li>Neumann, 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=Ubiquitinated+TDP-43+in+Frontotemporal+Lobar+Degeneration+and+Amyotrophic+Lateral+Sclerosis.">Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis.</a> Science. 314 (5796), 130 (2006).</li><li>Bishof, I., Diner, I., Seyfried, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Intrinsically+Disordered+Low+Complexity+Domain+is+Required+for+U1-70K+Self-association.">An Intrinsically Disordered Low Complexity Domain is Required for U1-70K Self-association.</a> FASEB J. 29 (Suppl 1), (2015).</li><li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+stageing+of+Alzheimer-related+changes.">Neuropathological stageing of Alzheimer-related changes.</a> Acta Neuropathol. 82 (4), 239-259 (1991).</li><li>Noguchi, A., 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=Isolation+and+Characterization+of+Patient-derived,+Toxic,+High+Mass+Amyloid+&#946;-Protein+(A&#946;)+Assembly+from+Alzheimer+Disease+Brains.">Isolation and Characterization of Patient-derived, Toxic, High Mass Amyloid &#946;-Protein (A&#946;) Assembly from Alzheimer Disease Brains.</a> J. Biol. Chem. 284 (47), 32895-32905 (2009).</li><li>Bai, B., 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=U1+small+nuclear+ribonucleoprotein+complex+and+RNA+splicing+alterations+in+Alzheimer's+disease.">U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.</a> Proc. Natl. Acad. Sci. 110 (41), 16562-16567 (2013).</li><li>Klunk, W. E., Pettegrew, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+&#946;-Amyloid+Protein+Is+Covalently+Modified+when+Dissolved+in+Formic+Acid.">Alzheimer's &#946;-Amyloid Protein Is Covalently Modified when Dissolved in Formic Acid.</a> J Neurochem. 54 (6), 2050-2056 (1990).</li><li>Yang, L. -. S., Gordon-Krajcer, W., Ksiezak-Reding, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+Released+from+Paired+Helical+Filaments+with+Formic+Acid+or+Guanidine+Is+Susceptible+to+Calpain-Mediated+Proteolysis.">Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain-Mediated Proteolysis.</a> J Neurochem. 69 (4), 1548-1558 (1997).</li><li>Bai, B., 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=U1+small+nuclear+ribonucleoprotein+complex+and+RNA+splicing+alterations+in+Alzheimer's+disease.">U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 110 (41), 16562-16567 (2013).</li><li>Hales, C. 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=U1+small+nuclear+ribonucleoproteins+(snRNPs)+aggregate+in+Alzheimer's+disease+due+to+autosomal+dominant+genetic+mutations+and+trisomy+21.">U1 small nuclear ribonucleoproteins (snRNPs) aggregate in Alzheimer's disease due to autosomal dominant genetic mutations and trisomy 21.</a> Mol Neurodegener. 9 (1), 15 (2014).</li><li>Xiong, L. -. W., Raymond, L. D., Hayes, S. F., Raymond, G. J., Caughey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+change,+aggregation+and+fibril+formation+induced+by+detergent+treatments+of+cellular+prion+protein.">Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein.</a> J Neurochem. 79 (3), 669-678 (2001).</li><li>Mirra, S. S., 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=The+Consortium+to+Establish+a+Registry+for+Alzheimer's+Disease+(CERAD).+Part+II.+Standardization+of+the+neuropathologic+assessment+of+Alzheimer's+disease.">The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease.</a> Neurology. 41 (4), 479-486 (1991).</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> Anal. Biochem. 150 (1), 76-85 (1985).</li><li>Guo, J. L., 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=Unique+pathological+tau+conformers+from+Alzheimer's+brains+transmit+tau+pathology+in+nontransgenic+mice.">Unique pathological tau conformers from Alzheimer's brains transmit tau pathology in nontransgenic mice.</a> J. Exp. Med. 213 (12), 2635-2654 (2016).</li><li>Matveev, S. V., 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=A+distinct+subfraction+of+A&#946;+is+responsible+for+the+high-affinity+Pittsburgh+compound+B-binding+site+in+Alzheimer's+disease+brain.">A distinct subfraction of A&#946; is responsible for the high-affinity Pittsburgh compound B-binding site in Alzheimer's disease brain.</a> J Neurochem. 131 (3), 356-368 (2014).</li></ol>

저자는 공개 없다.

여기 소개 하 고 다양 한 misfolding 분석 기능 단백질 질량 분석 기반 proteomics 분석에서까지 실험적인 응용 프로그램에 적용 하는 약식된 단일 단계 세제 분별 프로토콜 토론 그리고 서 부 럽입니다. 5 , 6 , 7 , 10 이 방법론은 아마도 가장 효과적인 치 매 등 신경 proteinopathies 공부 하는 데 사용 하는 경우, ?...

Alzheimer의 질병 (광고), 파 킨 슨 병 (PD), 헌팅턴의 질병 (HD), 루 경화 증 (ALS), 밀접 하 게 관련된 프리온 질환 등 신경 퇴행 성 질환은 proteinopathies의 점진적인 축적에 의해 특징 세제 불용 성 단백질 집계 동반 인지 뇌에서 떨어진다. 1 , 2 이 공유 병 적인 기능 이상 이러한 신경 퇴행 성 질환의 병 인에 중심 역할을 담당할 생각 이다. 2 이러한 집계 일반적으로 β크로스 misfolded 단백질 전시의 단위를 반복으로 구성 된 고분자 녹말 체 섬유의 구성-구조. 1 , 2 , 3 , 4 화학적, 녹말 체 집계는 화학 또는 열 변성을 가용 화, 그들의 정화, 분석에 독특한 도전을 선물 한다3 높은 저항 하 고 전통적인 생 화 확 적인 기술을 통해 공부. 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 unsurprisingly, 세제 불용 성 단백질 분수가 되었습니다 misfolded 단백질의 축적을 포함 하는 신경 퇴행 성 질환의 이상으로 많은 연구의 초점. 6 , 12 , 13 , 14
생 화 확 적인 분류 기법 종종 사후 뇌 homogenates에서 세제 불용 성 부분을 풍부 하 게 이용 되어 있다. 6 , 12 , 13 , 14 가장 일반적인 방법 중 하나는 버퍼와 조직 homogenates의 연속 추출과 엄중, 가용성과 불용 성 부분을 분할 하는 ultracentrifugation 다음 증가의 세제 포함 됩니다. 일반적으로 사용 되는 순차 분류 프로토콜6,14 세제 무료 낮은 소금 (LS) 버퍼에서 냉동된 조직 샘플의 균질 화를 포함 하 고 결과 불용 성 펠 릿은 다음 순차적으로 추출 높은 소금, 비 이온 세제, 높은 자당을 포함 하는 버퍼, 이온 세제 그리고 마지막으로 chaotropes 요소 처럼. 6 , 14 이러한 순차적 분별 프로토콜의 명백한 결점은 상당한 시간과 노동 투입을 완료 하는 데 필요한. 균질과 ultracentrifugation를 포함 하 여 전형적인 5 단계 분류 프로토콜 걸릴 수 있습니다 몇 시간 또는 일. 4 , 6 , 7 , 10 , 15 , 16 , 17 , 18 또한, 많은 pathologic 단백질 집계 남아 모든 가혹한 조건19,20 하지만 불용 성 생성 된 분수의 대부분은 제한 된 값의. 따라서, 높은 소금 농도 및 비 이온 세제 덜 엄격한 분류 단계 크게 중복 됩니다.
이전 연구는 이온 세제 N-라우릴-sarcosine (sarkosyl)는 단순화 된 단일 단계 세제 분별 프로토콜에 대 한 우수한 후보 나타났습니다. 5 , 6 , 12 , 13 , 14 , 21 , 22 , 23 변성 세제로 sarkosyl는 충분히 solubilizing misfolded 단백질 집계 베타-아 밀 로이드 (Aβ),6,11의 구성 없이 뇌에서 기본적으로 접힌된 단백질의 대부분을 solubilize 엄격한 phosphorylated 타우 (pTau),6 타르 DNA 묶는 단백질 43 (TDP-43),14 알파 synuclein,12,13 scrapie,23 또는 u 1 작은 핵 ribonucleoproteins (U1 snRNPs) u 1-70 K 등. 5 , 21 , 22 Sarkosyl 유비 쿼터 스 음이온 세제 나트륨 라우릴 황산 염 (SDS) 보다 덜 엄격으로, SDS 처리를 견딜 수 없는 misfolded 단백질 집계의 덜 강력한 oligomeric 형태 유지 합니다. 9
이전에 우리는 더 힘 드는 순차 분류 방법론에 유사한 결과 달성 하는 약식된 세제 분별 프로토콜을 설명 합니다. 5 덜 엄격한 분류 단계를 생략 하 여 우리 수 있었다 사후 인간의 뇌에서 세제 불용 성 단백질의 농축에 대 한 손쉬운 단일 단계 분류 프로토콜을 개발. 5 여기에 설명 된이 상세한 프로토콜 서쪽에 게 더 럽 히기에서 배열 하는 실험적인 응용 프로그램의 광범위 한 적합 하 고 기능적인 단백질 misfolding 및 집계 시드를 질량 분석 기반 단백질 분석 실험. 5 , 6 , 21

<table><tbody><tr><td>Protease and phosphatase inhibitor cocktail, EDTA-free (100X)</td><td>Thermo Fisher</td><td>78441</td><td>protease &amp;amp; phosphatase inhibitor cocktail</td></tr><tr><td>Sonic Dismembrator System (ultrasonicator)</td><td>Fisher Scientific</td><td>FB505110</td><td>microtip ultrasonicator</td></tr><tr><td>Optimax TLX Ultracentrifuge</td><td>Beckman Coulter</td><td>361545</td><td>refrigerated ultracentrifuge</td></tr><tr><td>TLA120.1 rotor</td><td>Beckman Coulter</td><td>362224</td><td>ultracentrifuge rotor</td></tr><tr><td>500 ul (8 x 34 mm) polycarbonate tubes, thickwall</td><td>Beckman Coulter</td><td>343776</td><td>ultracentrifuge tubes for TLA120.1 rotor</td></tr><tr><td>4X SDS sample buffer</td><td>Home-made</td><td>N/A</td><td>SDS-PAGE</td></tr><tr><td>TCEP solution, neutral pH</td><td>Thermo Fisher</td><td>77720</td><td>reducing agent</td></tr><tr><td>(TBS) blocking buffer</td><td>Thermo Fisher</td><td>37542</td><td>blocking buffer</td></tr><tr><td>(TBS) blocking buffer + 0.05% Tween 20</td><td>Thermo Fisher</td><td>37543</td><td>blocking buffer and antibody diluent</td></tr><tr><td>4-12% Bolt Bis-Tris Plus gels, 10-well</td><td>Thermo Fisher</td><td>NW04120BOX</td><td>precast SDS-PAGE gels</td></tr><tr><td>MES SDS Running Buffer (20X)</td><td>Thermo Fisher</td><td>B0002</td><td>SDS-PAGE running buffer</td></tr><tr><td>N-Lauroylsarcosine sodium salt (sarkosyl)</td><td>Sigma Aldrich</td><td>L5777-50G</td><td>detergent</td></tr><tr><td>Anti-Tau-2 (pan tau) antibody</td><td>Chemicon</td><td>MAB375</td><td>antibodies</td></tr><tr><td>Anti-phospho-threonine 231 Tau antibody</td><td>Millipore</td><td>MAB5450</td><td>antibodies</td></tr><tr><td>Anti-phospho-seroine 202 and threonine 205 Tau antibody (AT8)</td><td>Thermo Fisher</td><td>MN1020</td><td>antibodies</td></tr><tr><td>Anti-early endosome antigen 1 (EEA1) antibody</td><td>abcam</td><td>ab2900</td><td>antibodies</td></tr><tr><td>Alexa Fluor 680 goat anti-mouse IgG (H+L) secondary antibody</td><td>Thermo Fisher</td><td>A21058</td><td>antibodies</td></tr><tr><td>Alexa Fluor 790 donkey anti-rabbit IgG (H+L) secondary antibody</td><td>Thermo Fisher</td><td>A11374</td><td>antibodies</td></tr><tr><td>iBlot2 Dry Blotting System</td><td>Thermo Fisher</td><td>IB21001</td><td>Gel transfer</td></tr><tr><td>iBlot2 Transfer Stacks, Nitrocellulose, mini</td><td>Thermo Fisher</td><td>IB23002</td><td>Gel transfer</td></tr></tbody></table>

enrichment of detergent-insoluble protein aggregates from human postmortem brain

이 연구에서 우리는 인간의 사후 뇌에서 세제 불용 성 단백질의 농축에 대 한 약식된 단일 단계 분류 프로토콜을 설명합니다. 이오니아 세제 N-라우릴-sarcosine (sarkosyl)는 효과적으로 뇌 조직 신경 proteinopathies의 넓은 범위에서 세제 불용 성 단백질의 농축을 같이 허용에 기본적으로 접힌된 단백질 solubilizes Alzheimer의 질병 (광고), 파 킨 슨 병 및 루 경화 증, 그리고 프리온 질병 인간의 제어 및 광고 사후 뇌 조직 되었고 무 균 sedimented ultracentrifugation sarkosyl 세제 불용 성 단백질 집계 pathologic phosphorylated 타우, neurofibrillary의 핵심 구성 요소를 포함 하 여 풍부 하 게 존재 하 여 광고에 tangles입니다. 서 부 럽 집계 phosphorylated 타우와 세제 수용 성 단백질, 제어 및 광고 두뇌에서 초기 Endosome 항 원 1 (EEA1)의 차동 가용성을 설명 했다. Proteomic 분석은 또한 β의 농축을 밝혀-아 밀 로이드 (Aβ), 타우, snRNP70 (U1-70 K), 그리고 apolipoprotein E (APOE) 제어, 이전 조직 분류 전략 일치의 그들에 비교 하는 광고 두뇌의 sarkosyl-불용 성 분수에 . 따라서,이 간단한 농축 프로토콜에 이르기까지 서 부 럽 고 기능 단백질에서 공동 집계 분석 실험 단백질 질량 분석 기반 실험 응용 프로그램의 광범위 한 이상적입니다.

축약된 한 단계 sarkosyl-분별 프로토콜 제어 및 광고 사후 뇌 (그림 1)에서 세제 불용 성 단백질 집계를 풍부 하 게 사용 되었다. TH-S, S1, S2, P2 분수에서 단백질 SDS-PAGE, Coomassie 파란 통 버퍼 Coomassie 화려한 블루 G-250 얼룩 버퍼와 부드러운 얼룩 뒤에 15 분에 대 한 고정 해결 했다. 때문에 s 2 분수에 단백질의 탐지 수준 물의 resuspension 단계는 선택 사항 (1.1.1...

Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

인간의 사후 뇌에서 세제 불용 성 단백질의 농축에 대 한 약식된 분류 프로토콜 설명 되어 있습니다.

인간의 사후 뇌에서 세제 불용 성 단백질의 농축

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

enrichment-detergent-insoluble-protein-aggregates-from-human

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Biochemistry

12.0K 조회수.  Emory School of Medicine. 인간의 사후 뇌에서 세제 불용 성 단백질의 농축에 대 한 약식된 분류 프로토콜 설명 되어 있습니다.

비디오: Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

인간의 두피 조직에서 Aβ Multimers의 SDS-PAGE/Immunoblot 감지는 항원 에피토프 검색을 사용 Homogenates

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

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신경과학

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

보통 인간의 뇌에 용해와 불용성 PRP의 Oligomers의 절연

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

의학

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

인간의 사후 뇌 조직에서 핵의 연결을 격리

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

생물학

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

합의 뇌 유래 단백질, 2D-DIGE 미니 2DE 면역 블롯을 모두 사용하여 인간과 쥐과 뇌 프로테옴 연구를 위해 추출 프로토콜

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

파킨슨 뇌에서, 수용성 알파 시누 클레인의 순차 추출

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

설치류 및 인간의 뇌 조직에서 액틴 중합 상태를 평가하기위한 시간 효율적인 형광 분광 법 분석 기반 분석

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). Collectively, these Nups function to regulate genome organization, gene expression, and nucleocytoplasmic transport (NCT). Recently, defects in NCT and alterations to specific Nups have been identified as early and prominent pathologies in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer&#39;s Disease (AD)/Frontotemporal Dementia (FTD), and Huntington&#39;s Disease (HD). Advances in both light and electron microscopy allow for a thorough examination of sub-cellular structures, including the NPC and its Nup constituents, with increased precision and resolution. Of the commonly used techniques, super-resolution structured illumination microscopy (SIM) affords the unparalleled opportunity to study the localization and expression of individual Nups using conventional antibody-based labeling strategies. Isolation of nuclei prior to SIM enables the visualization of individual Nup proteins within the NPC and nucleoplasm in fully and accurately reconstructed 3D space. This protocol describes a procedure for nuclei isolation and SIM to evaluate Nup expression and distribution in human iPSC-derived CNS cells and postmortem tissues.

This protocol describes an optimized workflow for nuclei isolation and super-resolution structured illumination microscopy to evaluate individual nucleoporins within the nucleoplasm and NPCs in induced pluripotent stem cell derived neurons and postmortem human tissues.

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

뇌에서 아밀로이드 피브릴 코어의 생화학 적 정제 및 프로테오믹 특성화

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Isolation of Amyloid Fibrils from Brain Tissue Extract

Source: Upadhyay, A. et al., <a href="https://app.jove.com/v/63816">Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.</a> J. Vis. Exp. (2022)
This video demonstrates the protocol for isolating&#160; amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

뇌 조직 추출물에서 아밀로이드 섬유의 분리

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

Isolation of Detergent-Insoluble Protein Aggregates From Human Postmortem Brain Tissue

Source: Diner, I., et al. <a href="https://app.jove.com/v/55835">Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain.</a> J. Vis. Exp. (2017).
This video explains the process of isolating and enriching detergent-insoluble protein aggregates from frozen postmortem human brain tissue. It demonstrates key steps, including homogenization, detergent treatment, sonication, and ultracentrifugation to separate and purify the insoluble aggregates. The final step involves solubilizing the aggregates in urea buffer for further analysis.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.&#160;&#160;
1. Homogenization and ...