<html> 1. 효소 deglycosylation <ol><li> 증발에 의한 수분 손실을 최소화하기 위해 PCR 튜브를 사용하십시오. 튜브 1 7 라벨 한 세트. </li><li> 10X G7 버퍼, 10X 당단백질 denaturing 버퍼, 그리고 10 % NP - 40을 녹여 부드럽게 내용을 혼합하기 위해 튜브를 누릅니다. 상온에서 보관. </li><li> 얼음에 효소 함유 튜브를 삽입합니다. 해동 / 정지 사이클을 최소화하기 위해보십시오. </li><li> DH 2 O 600 μl에 hCGβ 유리병 (150 μg)의 내용을 해산하고 얼음에 보관. </li><li> DH 2 O.에서 10X 주식을 diluting하여 1X G7 버퍼 1 ML 준비 </li><li> 25 μl 1X G7 버퍼에 PNGase F 0.5 μl를 희석하여 얼음 계속. </li><li> 2 μl α - N - Acetylgalactosaminidase 각, α1 - 2 Fucosidase, β1 - 3 갈락토 및 α1 - 3, 6 갈락토를 결합하여 exoglycosidase 믹스 (예 혼합) 준비합니다. </li><li> 표시된로 PCR 튜브를 설정합니다 : </li></ol>AK &quot;&gt; <table border="1"><tbody><tr><td> 튜브 / 샘플 # </td><td> 일 </td><td> 이 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> hCGβ (0.25mg/ml) </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> - </td><td> - </td><td> - </td></tr><tr><td> 10X 당단백질는 버퍼 Denaturing </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td></tr><tr><td> DH 2 O </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 9μl </td><td> 9μl </td><td> 9μl </td></tr></tbody></table><ol start="9"><li> 튜브를 대장,에 부드럽게 믹스 및 장소thermocycler는 뚜껑을 닫고 94시 10 분 잠복기하여 단백질을 변성 ° 4 ° C 개최 다음 C (thermocycler에서 PCR 튜브를 사용하여 크게 소량의 샘플의 증발을 방지). </li><li> thermocycler에서 튜브를 제거하고 눈에 보이는 결로를 제거하는 원심 분리기. </li><li> 표시된대로 다음과 같은 시약을 추가합니다 : </li></ol><table border="1"><tbody><tr><td> 샘플 # </td><td> 일 </td><td> 이 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> 10% NP - 40 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td></tr><tr><td> 10X G7 버퍼 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5 &amp; 뮤; 리터 </td><td> 2.5μl </td></tr><tr><td> PNGase F 1시 50분 dil. </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td></tr><tr><td> Deglycosylation 믹스 </td><td> - </td><td> - </td><td> 2μl </td><td> 2μl </td><td> - </td><td> 2μl </td><td> 2μl </td></tr><tr><td> EG 믹스 </td><td> - </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td></tr><tr><td> DH 2 O </td><td> 10μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td></tr><tr><td> 총 반응 권. </td><td> 25 μ의 리터 </td><td> 25 μ의 리터 </td><td> 25 μ의 리터 </td><td> 25 μ의 L &lt;/ TD&gt; <td> 25 μ의 리터 </td><td> 25 μ의 리터 </td><td> 25 μ의 리터 </td></tr></tbody></table><ol start="12"><li> 새로운 모자를 (그들은 하나의 인큐베이션 사이클 후에 제대로 맞지 않기 때문에 사용하는 것들을 버리고)를 사용하여 PCR 튜브를 닫습니다. </li><li> 부드럽게 4 번 감청 다음 내용을 아래 스핀하여 튜브를 섞는다. </li><li> 4 시간 ° C가 다음 4 ° C.에 샘플을 냉각 37 thermocycler과 부화에 튜브를 삽입 </li></ol> 2. deglycosylated 샘플 SDS - PAGE <ol><li> 1.25M DTT 4 μl를 추가하여 130 μl 신선한 배 감소 SDS 로딩 버퍼를 준비합니다. </li><li> 각 샘플에 대한 준비가 배 감소 SDS 로딩 버퍼의 12.5 μl를 추가합니다. </li><li> 새로운 모자와 튜브를 닫고 부드럽게 섞어 튜브를 누릅니다. </li><li> 94에서 thermocycler에 튜브를 품어 ° C 4 ° C.에 냉각 후 5 분에 대한 </li><li> 각 시료 30 μl와 10~20% 트리스 - 글리신 젤의 단백질 마커의 10 μl를로드합니다. 일부 3.1 예제의 나머지 부분을 저장합니다. ColorPlus Prestained 단백질 마커의 10μl를로드합니다. </li><li> 염료 앞에는 젤 하단 때까지 실온에서 130 볼트에서 젤 Electrophorese. </li><li> 젤이 실행 완료되면, 캐스트에서 젤을 제거하고 충분한 Coomasie 파란 젤을 충당하기 위해 얼룩이있는 작은 플라스틱 상자에 넣습니다. </li><li> 부드러운 교반 1 시간 동안 젤 얼룩. </li><li> destain 솔루션의 50 ML 30 분 동안 겔 세 번 씻으십시오. </li><li> 흰색 라이트 transilluminator 또는 스캐너를 사용하여 이미지를 기록합니다. 또는 젤은 프레임에 셀로판의 시트 사이에 건조하실 수 있습니다. </li></ol> 3. SDS - PAGE의 젤의 glycosylated 단백질의 검출을위한 프로 Q 에메랄드 300 <ol><li> 2.1 겔)과 병행하여, 10-20% 트리스 - 글리신 젤에있는 샘플의 나머지를로드합니다. 로드 10과 뮤; ColorPlus Prestained 단백질 마커의 리터. </li><li> 젤 DMF와 프로 - Q 에메랄드 시약을 분해 및 재고 수정, 워시를 준비 실행하고 프로 - Q 에메랄드 300 솔루션을 산화하는 동안 키트와 함께 제공된 제품 설명서를 다음과 얼룩. </li><li> 전기 영동이 완료되면 캐스팅에서 젤을 제거하고 플라스틱 상자에 넣습니다. </li><li> 수정 솔루션 100 ML을 추가하고 부드러운 동요와 함께 실온에서 하룻밤을 떠나는하여 겔을 수정. </li><li> 이방인의 교반과 함께 상온에서 10-20분에 대한 씻으 솔루션의 100 ML로 젤을 씻으십시오. 신선한 씻어 솔루션 세척을 반복합니다. </li><li> 솔루션을 산화의 25 ML 30 분 부드러운 동요로 젤을 잠복기로 탄수화물을 산화. </li><li> 3.5 단계에서 설명한대로 젤 씻으십시오. </li><li> 젤은 25 ML 단계 3.2에서 녹아 프로 - Q 에메랄드 300 시약 용액 500 μl를 추가하여 신선한 프로 - Q 에메랄드 300 얼룩을 준비 세척하는 동안키트에 제공된 버퍼를 염색법. </li><li> 3.8 단계에서 준비한 얼룩의 25 ML을 추가하고 90-120분위한 부드러운 동요와 함께 어둠 속에서 잠복기로 젤 얼룩. </li><li> 3.5 단계에서 설명한 두 세척 단계를 반복합니다. </li><li> 300nm에서 UV transilluminator로 이미지를 기록합니다. prestained 사다리를 보여주는 젤의 하얀 빛을 그림에 UV 이미지를 겹쳐 프로 - Q 에메랄드 그린으로 표시되는 80 kDa의 마커를 사용합니다. </li><li> 3.11 단계의 프로 - Q 에메랄드 묻은 젤로 단계 2.10에서 Coomasie 스테인드 젤의 이미지를 비교합니다. </li></ol> 4. 대표 결과 효소 단백질 deglycosylation 후 마이 그 레이션의 변화는 그림 2에 표시됩니다. PNGase의 F 처리 (N - glycans의 제거, 차선 2), 그리고 Deglycosylation 믹스 (; 플러스 엔도와 O - glycans를 제거하는 exoglycosidases, 레인 3 PNGase F)와 (과 패널, 레인 1) 컨트롤 샘플을 비교합니다. 더 이상 감소하지크기 추가 glycosidases (레인 4) 소화 후 볼 수 있습니다. glycans가 제거 등 대량의 변경 외에도 밴드가 선명된다. 17 kDa의 마커 (별표)에서 실행 밴드는 완전히 deglycosylated hCGβ polypeptide (: 16 kDa MW)를 나타냅니다. 다른 밴드는 불완전 deglycosylation 또는 hCGβ 샘플에있는 여러 미확인 단백질 (레인 1 참조)에서 파생 수도 있습니다. 레인 5-7가 (컨트롤) glycosidases에 해당하는 밴드를 보여줍니다. 에메랄드 그린으로 당단백질의 얼룩이 시약은 단백질 분자에있는 모든 glycans를 산화 및 얼룩 패널 B.에 표시됩니다. hCGβ이 효소 (차선 4 차선 1) deglycosylated대로 따라서 신호의 강도가 줄어 듭니다. 레인 3과 4에서 잔여 신호가 사용되는 효소에 강한 glycan 모티프의 존재를 나타냅니다. 단백질 마이 그 레이션은 동일하지만 : 레인 4에 사용되는 추가 glycosidases 몇 가지 추가 설탕 잔류물을 제거얼룩의 강도에서 약간의 감소를 볼 수 있습니다. 저항 설탕 moieties은 모든 단백질 종에 존재되지 않은 몇 가지 밴드는 그들이 광범위 deglycosylated되었습니다 나타내는 에메랄드 그린 (괄호에 UV 이미지 결석)에 의해 감지되지 않았습니다. 추가 데이터 hCGβ가 glycosylated heterogeneously되는 결론을 지원합니다. 레인 2 위 밴드가 많은 glycans 그룹이 아직 존재했음을 나타냅니다 밝은 상태에서 차선 2 (화살표)에 대한 낮은 밴드는 에메랄드 그린 이미지에 희미합니다. 이 데이터는 마우스 세포에서 표현 재조합 hCGβ 여러 glycoforms에게 9에 들어있는 결론을 지원합니다. 심지어 같은 단백질에 다른 수는 없지만 이러한 다양한 glycoforms 일부 polypeptides 각 합의 사이트 및 / 또는 일부 glycans에서 glycan을받지 못한 글리코 실화의 고유 이질로 인해 아르가 연장됩니다. <img alt="그림 1" src="/files/ftp_upload/3749/3749fig1.jpg" /> 그림 1.분비 또는 세포 표면 glycoproteins에 대한 일반적인 글리코 실화 패턴. <img alt="그림 2" src="/files/ftp_upload/3749/3749fig2.jpg" /> hCGβ의 효소 deglycosylation을 보여주는 그림 2. SDS - PAGE의 젤. 패널 B가 glycosylated 단백질의 시각화를위한 프로 - Q 에메랄드 300의 결과를 보여주는 동안 패널 A는 Coomasie 파란 얼룩을 보여줍니다. 샘플 번호 : 1, hCGβ 제어, 2, PNGase F 소화, 3, Deglycosylation 믹스 소화, 4, Deglycosylation 믹스 플러스 exoglycosidases의 소화, 샘플 5-7이 (O - Glyc, O - Glycosidase 시약 컨트롤되며 GNA, β - N - Acetylglucosaminidase; 걸 / Fuc, β1 - 3 갈락토, α1 - 3, 6 갈락토 및 α1 - 2 Fucosidase, GalNA, α - N - Acetylglalactosaminidase, NEU, Neuraminidase, PNGase, PNGase의 F)

<ol>
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저자는이 문서에서 사용되는 시약의 많은 생산 뉴잉글랜드 Biolabs로 고용됩니다.

glycan 특정 시약은 데이터의 해석을 용이하게하면서 방법은 관심의 단백질의 글리코 실화 상태에 대한 유용한 정보를 제공할 수있는 효소 deglycosylation와 SDS - PAGE를 사용하여 여기에 설명했다. 이 프로토콜은 단백질 글리코 실화의 초기 연구를위한 것입니다 그리고 그것은 특히 포유 동물 세포에서 분비 및 멤브레인 glycoproteins에 적합합니다 :이 경우에는 선택한 효소는 특히 모든 N - glycans을 제거하고 또는 N - glycans 플러스 및 가장 일반적인 설탕 것입니다 확장하고 O - glycans의 핵심을 형성. Glycosidases은 당분과 단백질 백본 모두의 무결성을 보존, 화학 deglycosylation 방법에 비해 온화한되는 추가 이점이 있습니다. 인원의 비율 (아미노산가 glycosylated있는), 글리코 실화의 정도, 또는 glycans의 미세 구조와 같은 m와 같은보다 정교한 기술을 결정을 명료하게하다하기엉덩이 분광법, 액체 크로마 토그래피 또는 NMR이 필요합니다. 간단 때문에,이 프로토콜의 여러 단계, 조정 대체, 그리고 / 또는 다양한 실험 요구를 수용하기 위해 결합 수 있습니다. 그러나 명확하게 해석될 수 결과를 얻기 위해서는 자사의 강점과 한계를 이해하는 것이 중요합니다. 첫째, glycosidases의 특이성과 순결이 중요한 : 프로 테아제 및 기타 오염 활동의 자유를 테스트에만 잘 특징 효소를 사용해야합니다. 불행하게도, glycosidases에 대한 표준 효소 단위 정의가 없으며 사용자가 제조 업체의 사양에 따라 적절한 대체를 결정한다. 둘째, 감지주의 선택이 필요합니다 :) 단백질 얼룩의 시약이 유용한 경우에만 분자 덩어리에 큰 변화에 deglycosylation 결과하십시오. 여기에 그림과 같은 명확한 결과는 항상 얻을 수 없습니다. 다른 경우에, 우리는 비정상적인 미를 보지 못했습니까deglycosylation 후 gration (멀리 예측 분자량, 심지어 느린 마이 그 레이션에서). 이러한 현상은 잘 이해 아니라, 그것은 이전에 조금이라도 변화가 단백질이 deglycosylated되었다는 증거입니다. B) 항체와 설탕 감지가 적용을 제한 독특한 과제를 제시라고 할 수 있습니다. 그것은 일반적인 안티 glycan 항체를 생성하는 것은 매우 어려운되었습니다, 그들은 대개 자신의 사용을 제한 복잡한 glycan 대상, 반대 사육하고 있습니다. 또한, 여러 단클론 항체 안티 - glycan이 원하지 않는 교차 반응 10을 표시합니다. C) Lectins이 (내장 설탕 선호도와 단백질) 잘 설탕 탐지에 적합합니다, 게다가, 그들은 glycan 구조에 대한 통찰력을 제공합니다. 그러나 모든 이들은 좁은 특이성을 가지고, 그리고 많은 사람들이 부분적으로만 (그들이 알 수없는 동질성을 가지고 있다고 의미) 특징입니다. 그 결과 긍정적인 렉틴의 얼룩은 GI의 존재의 표시가 아닌 증거를 제공으로이리와 설탕. D) 화학 라벨링 키트 (당분의 periodate 산화에 따라) 모든 glycoproteins을 얼룩이 선택의 방법이며, 따라서 잘 deglycosylation에 따라 적합. 알 수없는 시료를 처리하면 당단백질 컨트롤을 포함하는 것이 좋습니다. Fetuin는 즉시 사용할 수 N합니다 - O - 당단백질, chorionic gonadotropin은 (두 subunits)도 좋은 선택입니다. 소 혈청 알부민은 (BSA)은 대조군으로 사용할 수 있습니다. 그러나 일부 비 glycosylated 단백질이 높은 농도에서 사용 특히, 프로 - Q 에메랄드 300과 약간 반응 것을인지해야합니다. 이와 프로토콜에서 사용되는 prestained 단백질 마커와 같은 비 glycosylated 분자량 표준, 선명한 밴드를 표시하는 장점이 있습니다. 유일한 기회로 프로 - Q 에메랄드 300 반응이 단백질 (80 kDa) 중 하나입니다. 따라서, 사용자는 Invitroge에서 막대 사탕 지팡이로 대신 당단백질 표준 사다리를 실행할 수도 있습니다N. 마지막으로, nucleocytosolic glycoproteins (하나의 O - GlcNAc로 수정되는)의 간단한에서 - 겔 감지 특이성 제어 11 β - N - Acetylglucosaminidase 함께 단클론 항체의 사용이 가능합니다.이 방법의 설명이없는 것입니다 glycosidases은 세포에있는 많은 glycoproteins와 glycoconjugates 연구를위한 유용한 도구입니다이 문서의 범위,하지만 그것은 언급되어야합니다. 12 :; NCBI의 책장 (20301239 PMID NBK1908 책장의 ID)에서 구할 무료 온라인, 글리코 실화의 알려진 모든 형태의 포괄적인 치료는 &quot;Glycobiology의 기초&quot;의 두 번째 버전에서 찾을 수 있습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 인간 chorionic gonadotropin의 β </td><td> 시그마 알드리치 </td><td> C6572 </td><td></td></tr><tr><td> PCR 튜브 </td><td> VWR </td><td> 20170-004 </td><td></td></tr><tr><td> PCR Thermocycler </td><td> 응용 Biosystems </td><td> 4,359,659 </td><td></td></tr><tr><td> PNGase F </td><td> 뉴 잉글랜드 Biolabs </td><td> P0704 </td><td> 버퍼와 함께 제공된 </td></tr><tr><td> 단백질 Deglycosylation 믹스 </td><td> 뉴 잉글랜드 Biolabs </td><td> P6039 </td><td> 버퍼와 함께 제공된 </td></tr><tr><td> α - N - Acetylglalactosaminidase </td><td> 뉴 잉글랜드 Biolabs </td><td> P0734 </td><td> 버퍼와 함께 제공된 </td></tr><tr><td> α1 - 2 Fucosidase </td><td> 뉴 잉글랜드 Biolabs </td><td> P0724 </td><td> 버퍼와 함께 제공된 </td></tr><tr><td> α1 - 3, 6 갈락토 </td><td> 뉴 잉글랜드 Biolabs </td><td> P0731 </td><td> 버퍼와 함께 제공된 </td></tr><tr><td> β1 - 3 갈락토 </td><td> 뉴 잉글랜드 Biolabs </td><td> P0726 </td><td> 버퍼와 함께 제공된 </td></tr><tr><td> G7 10X 버퍼 (500 MM의 인산 나트륨) </td><td> 뉴 잉글랜드 Biolabs </td><td> --- </td><td> PNGaseF 또는 Degl 믹스와 함께 제공된 </td></tr><tr><td> 10X 당단백질 Denaturing 버퍼 (5 % SDS, 400 MM DTT) </td><td> 뉴 잉글랜드 Biolabs </td><td> --- </td><td> PNGaseF 또는 Degl 믹스와 함께 제공된 </td></tr><tr><td> 10% NP - 40 </td><td> 뉴 잉글랜드 Biolabs </td><td> --- </td><td> PNGaseF 또는 Degl 믹스와 함께 제공된 </td></tr><tr><td> 배 SDS 로딩 버퍼 (187.5 MM 트리스 - HCL 산도 6.8, 6% SDS, 30 % 글리세롤, 0.03 % 브롬ophenol 블루) </td><td> 뉴 잉글랜드 Biolabs </td><td> B7703 </td><td> 1.25M DTT와 함께 제공된 </td></tr><tr><td> ColorPlus 단백질 마커를 Prestained </td><td> 뉴 잉글랜드 Biolabs </td><td> P7709 </td><td></td></tr><tr><td> 10-20% 트리스 - 글리신 Multigel </td><td> 코스모 바이오 (주) </td><td> DCB - 414893 </td><td></td></tr><tr><td> 카세트 전기 단위 </td><td> 코스모 바이오 (주) </td><td> DCB - 303111 </td><td></td></tr><tr><td> 전기 전원 공급 장치 EPS 301 </td><td> GE 헬스케어 </td><td> 18-1130-01 </td><td></td></tr><tr><td> 프로 - Q 에메랄드 300 당단백질 얼룩 키트 </td><td> Invitrogen </td><td> P - 21857 </td><td></td></tr><tr><td> 똑똑 블루 R </td><td> 시그마 알드리치 </td><td> B0149 </td><td></td></tr><tr><td> AlphaImager HP 시스템 </td><td> 휴대폰 Biosciences </td><td> 92-13823-00 </td><td></td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

글리코 실화, covalently 연결된 당분의 추가는, 크게 이러한 세포 접착, 분자 거래, 통관 및 신호 전달 1-4로 프로세스에 영향을 미칠 수있는 단백질의 주요 포스트 translational 수정합니다. 또는 세린 또는 트레오닌 잔류물에 (O - 연결된) (그림 1) eukaryotes에서 분비 경로에서 가장 일반적인 글리코 실화의 수정 합의 아스파라긴의 잔류물 (N - 연결된)에 추가됩니다. 최종 제품은 다른 종, 티슈, 또는 단백질 중에서 크게 다를 수 있지만 N - glycan 합성의 개시는 매우, eukaryotes에 보존되어있다. 일부 glycans은 수정되지 않은 ( &quot;높은 mannose N - glycans&quot;) 또는 추가 잠돌군 Zz ( &quot;복잡한 N - glycans&quot;)로 처리됩니다 남아 있습니다. 광역의 다양성은 동물 세포의 일반적인 N - Acetylgalactosamine (GalNAc) 잔여물로 시작하지만, 낮은 생물 1 다를 O - glycans에 대한 발견이다. 엔트 &quot;&gt; 단백질의 글리코 실화의 상세한 분석은 그 자체에게로 필드이며 광범위한 자원과 전문성이 제대로 실행이 필요합니다. 삭제 당분 (glycosidases가)의 글리코 실화 상태에 대한 일반적인 생각을 가능하게 사용할 수 효소 그러나 다양한 . 표준 실험실 환경에서 단백질이 여기 모델 당단백질의 분석 glycosidases의 사용 설명 :. 2 N - glycans과 네 개의 O - glycans 5 운반 재조합 인간 chorionic gonadotropin 베타 (hCGβ) 기술은 단순이 필요합니다 계측 및 일반 소모품, 그리고 그것은 쉽게 여러 개의 당단백질 샘플의 분석을 적용할 수 있습니다. 여러 효소는 당단백질 연구를 병렬로 사용할 수 있습니다. PNGase F는 N - 연결된 glycans 6,7의 거의 모든 종류 제거할 수 있습니다. O - glycans 들어, 사용 가능한 효소가 없다고 다니엘이 일부터 손상 올리 고당 수E 단백질 백본. 대신, O - glycans 그러면 쉽게 O - Glycosidase에 의해 제거되는 짧은 핵심에 exoglycosidases으로 손질하고 있습니다. 단백질 Deglycosylation 믹스는 PNGase Neuraminidase F, O - Glycosidase (sialidase), β1 - 4 갈락토 및 β - N - Acetylglucosaminidase이 포함되어 있습니다. 그것은 동시에 N - glycans와 일부 O - glycans 8 제거하는 데 사용됩니다. 마지막으로, Deglycosylation 믹스는에있는 수 달리 강한 단당류를 제거하는 데 도움 다른 exoglycosidases의 혼합물 (6 갈락토, α1 - 3, α1 - 2 Fucosidase, α - N - Acetylgalactosaminidase 및 β1 - 3 갈락토)로 보충했습니다 특정 O - glycans. SDS-PAGE/Coomasie 파란색 glycosidase 처리 전후의 단백질 마이 그 레이션의 차이를 시각화하는 데 사용됩니다. glycans가 succe 있으므로 또한, 설탕 특정 얼룩 방법, ProQ 에메랄드 - 300는 신호 저하를 보여줍니다ssively 삭제되었습니다. 효소 deglycosylation가 필요에 따라 단백질의 큰 수량을 수용까지 확장할 수 있지만이 프로토콜은, 당단백질 (0.5-2 μg)의 소량의 분석을 위해 설계되었습니다.

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

SDS - PAGE에 의해 다음 glycoproteins에서 설탕을 제거하는 구체적인 glycosidases를 사용하면 단백질 시료에 glycan 변경을 탐지하는 유용한 방법이며 초기 glycobiology 연구를위한 좋은 선택입니다. deglycosylation 다음과 같은 변경 사항은 겔 이동성의 교대 ​​또는 glycan 민감한 시약과 염색법에 의해 감지 수 있습니다.

특정 엔도와 exoglycosidases를 사용하여 단백질 글리코 실화의 식별 및 특성

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

Research

JoVE Journal

Biology

36.4K 조회수.  New England Biolabs. SDS - PAGE에 의해 다음 glycoproteins에서 설탕을 제거하는 구체적인 glycosidases를 사용하면 단백질 시료에 glycan 변경을 탐지하는 유용한 방법이며 초기 glycobiology 연구를위한 좋은 선택입니다. deglycosylation 다음과 같은 변경 사항은 겔 이동성의 교대 ​​또는 glycan 민감한 시약과 염색법에 의해 감지 수 있습니다.

비디오: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

연구

생물학

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

탠덤 동질 정제를 사용하여 단백질 나누기 파트너의 식별

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

단지 샘플의 특정 단백질 글리코 실화의 다중 높은 처리량 프로파일을위한 화학을 차단 항체 Microarray

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

RIP 칩을 사용하여 세포 추출물에서 mRNAs, microRNAs 및 Ribonucleoprotein 단지의 단백질 구성 요소의 분리 및 확인에 방법

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

셀 침습적 DISC1 단백질 종의 생성, 정제 및 특성

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

미생물 식별 및 특성화를위한 Pyrosequencing

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

특정 게놈 지역과 enChIP으로 관련 분자의 식별의 분리

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.