Name: visualization of protein-protein interaction in nuclear and cytoplasmic fractions by co-immunoprecipitation and in situ proximity ligation assay
Uploaded: 2017-01-16T15:00:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay at JoVE.com

This study was supported by the Swiss National Science Foundation (SNSF, 31003A_163305).

1. 공동 면역 침전 <ol><li> 씨앗 하나 6 웰 플레이트에 웰 당 2 × 10 5 세포에서 HEK293 세포 2 ㎖의 10 % 소 태아 혈청 (FBS)와 함께 둘 베코의 수정 이글의 중간 (DMEM) 보충 100 U / mL의 페니실린 - 스트렙토 마이신 (펜 연쇄상 구균) . </li><li> 5 % CO 2, 37 ℃에서 48 시간 동안 세포를 성장. </li><li> 차가운 인산 완충 식염수 (PBS)로 3 회 세포를 씻으십시오. 4 ° C에서 5 분 500 XG에 원심 분리하여 수확 세포. </li><li> 상업 핵 및 세포질 추출 시약을 사용하여 핵 및 세포질 분수를 준비합니다. 일부 수정과 제조업체의 지시 사항을 따르십시오. <ol><li> 하나의 공동 면역 실험 (한 6 웰 플레이트의 3 우물에서 약 90 %의 컨 플루)에 대한 3 × 10 6 세포를 사용합니다. 최고 속도로 15 초 동안 300 μl의 차가운 세포질 추출 용액 (CER의 I)와 소용돌이를 추가합니다. </li><li> 얼음에 30 분 동안 품어. 배양, vorte 중5 초마다 10 분 동안 X. </li><li> 5 초 동안 16.5 μl의 차가운 세포질 추출 용액 II (CER II), 소용돌이를 추가하고 얼음에 5 분 동안 품어. </li><li> 4 ° C에서 5 분 동안 16,000 XG에 5 초 원심 분리기를위한 소용돌이. </li><li> 새로운 사전 냉각 튜브에 뜨는 (세포질 추출물)를 전송하고, 사용까지 얼음에 보관하십시오. </li><li> 때마다 최대 피펫 팅에 의해 5 배 아래로 1 ml의 차가운 PBS와 불용성 펠렛 (포함하는 핵)을 세 번 씻으십시오. 세척 후 PBS를 제거합니다. </li><li> 15 초 동안 150 μl의 차가운 핵 추출 용액 (NER), 소용돌이를 추가하고 얼음에 1 시간을 품어. 배양하는 동안, 15 초 및 피펫 200 μl의 팁 10 시간마다 10 분 동안 소용돌이. </li><li> 4 ℃에서 10 분 동안 16,000 XG에 15 초 동안 소용돌이와 원심 분리기. </li><li> 새로운 사전 냉각 튜브에 뜨는 (핵 추출물)로 이동시켜 사용할 때까지 얼음에 보관하십시오. </li></ol></li><li> 각각의 입력으로 핵 및 세포질 추출물의 볼륨의 10 %를 가져 가라. </li><li> 에이1 ml의 최종 용적에 남아있는 추출물을 차가운 PBS를 위해 dd. 얼음에 사용할 때까지 보관하십시오. 주 : 단백질 G 공역 자성 비드를 사용하는 경우 사전 클리어링이 필수적인 것은 아니다. 백그라운드가 높은 경우에는, 40 ㎕의 단백질 G 공역 자성 비드 (50 % 슬러리)와 회 전자에서 30 분 동안 4 ℃에서이 단계에서 해물 희석 한 용액을 배양하여 전 소거를 수행한다. 자기 랙 구슬에서 분리 된 상층 액 (사전 허가 해물). 구슬을 폐기하십시오. </li><li> 단백질 G-복합 자석 구슬 몇 차 항체로, 4 μg의 토끼 다 클론 항 RBM3 항체 또는 토끼의 IgG (음성 대조군) 200 ㎕의 PBS에 플러스 트윈 20 버퍼와 단백질 G - 복합 자석 구슬 (50 % 슬러리) μL (40)을 품어 (PBST, 0.02 % 트윈 20)가 회 40 분 동안 실온 (RT)에서. </li><li> 별도의 자기 랙 구슬과 뜨는 항체를 결합하고, 상층 액을 버린다. 200 μL PBST (0.02 %) 한 번 구슬을 씻으십시오. </li><li> 1 추가용액은 비드 해물을 (또는 필요한 사전 허가 해물 경우)로 희석하고, 밤새 4 ℃에서 회전에 배양한다. </li><li> 다음 날, 자기 랙에 의해 구슬과 상층 액을 분리하고, 상층 액을 버린다. 20 rpm의 고정 된 속도로 회 전자에 4 ° C에서 각각의 튜브 0.5 ml의 PBST (0.02 %)와 3 × 10 분을 씻으십시오. </li><li> 40 ㎕의 시료 완충액 (1X 상업용 샘플 버퍼의 50 mM 1,4- 디티 오 트레이 톨 (DTT))를 첨가하여 비드로부터 단백질을 용출. 10 분 동안 70 ℃에서 가열한다. 새로운 1.5 ML 튜브에 스핀 다운 및 전송 액체. </li><li> 10 분 동안 70 ℃에서 가열 (1X 상업용 샘플 완충액 50 mM의 DTT, 최종 농도)을 샘플 버퍼에 입력 해물을 희석. 프리 캐스트 4-12% 비스 - 트리스 젤 마지막 단계에서로드 입력과 면역 단백질. 각 웰의 적재 부피는 20 μl를 초과 할 수 없습니다. 35 분 동안 얼음에 1 배 상업 운전 버퍼에 전기 영동을 수행합니다. 참고 :로드 핵 및 세포질 추출물셀의 동일한 초기 량을 반영 (2) (V / V를) : 1의 비율로 S. </li><li> 4 ° C에서 2 시간 동안 30 V에서 1 배 상업 전송 버퍼에서 PVDF 막에 전달 단백질. </li><li> 5 % 차단 막을 진탕 기에서 40 분 동안 RT에서 우유 PBST (0.1 % 트윈 20)를 미끄러 져. </li><li> 밤새 4 ° C에서 PBST (0.1 %)에서 : 일차 항체와 멤브레인 (1,000 두 1에 희석) 품어. </li><li> 쉐이커에 RT에서 PBST (0.1 %)와 3 × 10 분을 씻으십시오. </li><li> 1 시간 동안 RT에서 PBST (0.1 %)에서, (5000 모두 1로 희석) 양 고추 냉이 퍼 옥시 다제 (HRP)와 함께 막을 인큐베이션 항 - 마우스 또는 항 - 토끼 이차 항체를 π 공역. </li><li> 쉐이커에 RT에서 PBST (0.1 %)와 3 × 10 분을 씻으십시오. </li><li> cm 2 막 당 0.2 ml의 강화 된 화학 발광 (ECL) 기판 혼합물을 사용하여, 5 분 동안 실온에서 품어. 빨간 불빛을 제외하고 이후이 단계에서 모든 빛을 피하십시오. </li><li> 액체 버리고 필름 카세트에 X 선 필름에 노출. 자동 필름 홍보를 사용하여 필름 개발ocessing 기계. </li></ol> 2. 면역 세포 화학 및 근접 내고 분석 <ol><li> 시드 0.4 ㎖의 DMEM 한 8 실, 폴리 D 라이신 코팅 된 슬라이드 챔버 당 1.5 × 104 세포의 HEK293 세포는 10 % FBS, 100 U / ㎖ 펜 연쇄상 보충. </li><li> 5 % CO 2, 37 ℃에서 48 시간 동안 세포를 성장. </li><li> 대기음 매체 및 RT에서 10 분 동안 4 % 파라 포름 알데히드 (PFA)로 세포를 고정한다. </li><li> 대기음 PFA, 챔버 당 0.5 ml의 PBS로 세척 3 × 10 분. </li><li> Permeabilize 하시려면 1 시간 동안 실온에서 PBS에서 0.5 % 트리톤 X-100, 5 % 정상 염소 혈청 (NGS)와 블록 셀. </li><li> 밤새 4 ° C에서 통에 PBS에서 0.1 % 트리톤 X-100 및 5 % NGS의 일차 항체와 함께 품어. 마우스 단일 클론 항 NF90 항체 토끼 다 클론 항 RBM3 항체 1을 희석 : PBS 100을 두 번 염색을 위해. 음성 대조군의 경우, 독립적으로 두 일차 항체 중 어느 하나를 생략하고, 제 3 제어 모두 항체 생략. 참고 : 광고 차단 및 희석 시약을 사용하지 마십시오. </li><li> 챔버 당 0.5 ml의 PBS로 3 × 10 분을 씻으십시오. </li><li> 면역 세포 또는 근접 내고 분석 프로토콜에 따라 <ol><li> 면역 세포 화학 <ol><li> 부화 1 : 500 희석 된 녹색 형광 염료가 결합 된 항 - 마우스 및 적색 형광성 염료 결합 항 - 토끼 이차 항체를 실온에서 1 시간 동안. 이후이 단계에서 빛을 피하십시오. </li><li> 10 분 동안 실온에서 PBS로 5,000 : 4 &#39;, 6-diamidin -2- phenylindol (DAPI)로 Counterstain과 핵 (1) 희석 하였다. </li><li> 챔버 당 0.5 ml의 PBS로 3 × 10 분을 씻으십시오. </li><li> 유리 슬라이드 건조에서 챔버를 제거합니다. 슬라이드 당 매체를 장착, 250 μ으로 마운트합니다. </li></ol></li><li> 근접 내고 분석 <ol><li> 근접 내고 분석 프로브를 준비합니다. 믹스와 일을 결찰 수있는 다른 올리고 뉴클레오티드로 연결된 두 개의 근접 내고 분석 프로브 (항 - 마우스 및 안티 - 토끼 보조 항체를 희석하나 8 챔버 슬라이드 (cm 2 당 약 40 ㎕를 320 ㎕의 총 부피로 PBS에서 0.1 % 트리톤 X-100 5 5 % NGS) 다른 두 연결 용액의 올리고 뉴클레오티드)을 모두 한 거친 첨가 . 실온에서 20 분 동안 인큐베이션. </li><li> 유리 슬라이드에서 챔버를 제거하고 희석 프로브를 추가 할 수 있습니다. 1 시간 동안 37 ℃에서 습도 인큐베이터에서 인큐베이션. </li><li> 연결 용액을 준비합니다. 8 리가 ㎕를 혼합 64 μl를 5 × 결찰 재고 및 248 μL의 H 2 O </li><li> 슬라이드에서 액체를 누른 1X 세척 버퍼 (A)에 2 × 5 분을 씻어 (키트와 함께 제공). </li><li> 라이 게이션 용액을 첨가하고 30 분 동안 37 ℃에서 습도 인큐베이터에서 배양한다. </li><li> 증폭 솔루션을 준비합니다. 이후이 단계에서 빛을 피 4 μL 중합 효소, 64 ㎕의 5 배 증폭 재고 및 252 μL의 H 2 O를 섞는다. </li><li> 슬라이드에서 액체를 누른 1X 세척 버퍼 A에 2 × 2 분을 씻어 </li><li> 앰프 추가lification 용액을 100 분 동안 37 ℃에서 어두운 습도 인큐베이터에서 배양한다. </li><li> 슬라이드에서 액체를 누른 1X 세척 버퍼 B에 2 × 10 분을 씻어 (키트와 함께 제공). 0.01x 세척 버퍼 B.에서 1 분 동안 세척 </li><li> 슬라이드를 건조 및 슬라이드 당 (DAPI)와 상업 장착 매체의 250 μl를 마운트. </li></ol></li></ol></li><li> 10 배의 접안 렌즈와 20 배 대물 렌즈를 사용하여 현미경 형광 신호를 검사합니다. CCD 카메라에 의해 화상을 취득. </li></ol>

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The authors have nothing to disclose.

두 방법에 대한 몇 가지 장점뿐만 아니라 단점이있다. 비교적 새로운 기술로서, 인접 결찰 분석법의 명백한 이점은 단일 세포 수준 대신 이기종 셀 배치의 단백질 - 단백질 상호 작용을 규명 할 수있는 가능성이다. 높은 크기 및 (공 초점 현미경으로 예) 해상도의 이미지는 하나의 형광 반점을 계산하여 정량화에 대한 가능성을 제공합니다. 대조적으로, 웨스턴 블랏과 공동 면역 침전 기술의...

핵 인자 90 (NF90)은 인터루킨 -2 후 전사 및 miRNA의 생합성 1-3의 규제의 바이러스 감염에 대한 반응 조절 등 다양한 기능을 가진 멀티 이소 단백질이다. RBM3는 RNA 결합 단백질 번역 및 miRNA의 생합성에 관여 저체온증 저산소증 4-6을 포함하는 다양한 스트레스에 의해 유도 될 수있다. 최근에, 우리는 단백질 복합체 7 NF90 및 RBM3를 발견했다. NF90 RBM3과의 상호 작용은 단백질 펼친 응답 7 단백질 키나제 RNA 같은 소포체 키나제 (PERK) 활성을 조절하는 것이 필수적이다. NF90 및 RBM3 모두 핵하지만 세포질에 작은 NF90의 비율과 RBM3 셔틀에 주로 위치하며, PERK 활동을 조절하는 특정 기능을 위해 서로가 예를 결합한다. 따라서, 어느 것을 나타낼 수 있고, 세포 내 구획에서-NF90 RBM3 상호 작용의 분포를 시각화하는 것이 중요각각의 구획에서의 다양한 역할. 수십 년 전에, 효모 두 하이브리드 (Y2H)는 두 단백질 (8) 사이의 상호 작용을 검출하기 위해 개발되었다. 그러나, 융합 단백질의 인공 건설, 거짓 양성 결과는이 방법의 적용을 제한하고있다. 오랫동안, 공동 면역 침전 특히 내인성 조건 9, 단백질 - 단백질 상호 작용을 분석하는 주요 기술이다. 최고 감도 및 정밀도가 요구되는 경우, 질량 스펙트럼에서 사용되는 동안에 공동 면역 침전 된 단백질 복합체를 분석, 웨스턴 블롯, 가장 편리한 기술이다. 최근, 근접 결찰 분석은 동일계 10,11 모두에서 세포 및 조직에서의 단백질 - 단백질 상호 작용을 검출하는 신규 한 방법으로 개발되었다. 여기서는 NF9 캡처에서 가장 인기있는 공동 면역 침전 법 및 비교적 신규 근접 결찰 분석 방법에 비해세포 내 분수에 0 RBM3 상호 작용. 우리는 또한 장점과 두 가지 기술의 한계를 논의했다.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Sigma</td><td>D6429</td><td>High glucose&lt;br/&gt; 4,500 mg/L</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco, Thermo Fisher Scientific</td><td>10270106</td><td></td></tr><tr><td>Penicillin-Streptomycin (PenStrep)</td><td>BioConcept</td><td>4-01F00-H</td><td></td></tr><tr><td>NE-PER Nuclear and Cytoplasmic Extraction Reagents</td><td>Thermo Fisher Scientific</td><td>78833</td><td></td></tr><tr><td>1,4-Dithiothreitol (DTT)</td><td>Carl Roth</td><td>6908.3</td><td></td></tr><tr><td>Dynabeads Protein G</td><td>Novex, Thermo Fisher Scientific</td><td>10003D</td><td></td></tr><tr><td>DRBP76 (NF90/NF110) antibody</td><td>BD Transduction Laboratories</td><td>612154</td><td>use 1:1,000 for WB and 1:100 for ICC/PLA</td></tr><tr><td>RBM3 antibody</td><td>ProteinTech</td><td>14363-1-AP</td><td>use 1:1,000 for WB and 1:100 for ICC/PLA</td></tr><tr><td>Lamin A/C antibody</td><td>Cell Signaling Technology</td><td>#2032</td><td>use 1:1,000 for WB&amp;nbsp;</td></tr><tr><td>anti-GAPDH antibody</td><td>Abcam</td><td>ab8245</td><td>use 1:1,000 for WB&amp;nbsp;</td></tr><tr><td>normal rabbit IgG</td><td>Santa Cruz</td><td>sc-2027</td><td></td></tr><tr><td>anti-rabbit IgG, HRP-linked secondary antibody</td><td>Cell Signaling Technology</td><td>#7074</td><td>use 1:5,000 for WB&amp;nbsp;</td></tr><tr><td>anti-mouse HRP secondary antibody</td><td>Carl Roth</td><td>4759.1</td><td>use 1:5,000 for WB&amp;nbsp;</td></tr><tr><td>Clarity Western ECL Blotting Substrate</td><td>Bio-Rad</td><td>#1705060</td><td></td></tr><tr><td>NuPAGE Novex 4-12% Bis-Tris Gel</td><td>Novex, Thermo Fisher Scientific</td><td>NP0321BOX</td><td></td></tr><tr><td>NuPAGE LDS Sample Buffer (4x)</td><td>Novex, Thermo Fisher Scientific</td><td>NP0007</td><td></td></tr><tr><td>NuPAGE MES SDS Running Buffer (20x)</td><td>Novex, Thermo Fisher Scientific</td><td>NP0002</td><td></td></tr><tr><td>NuPAGE Transfer Buffer (20x)</td><td>Novex, Thermo Fisher Scientific</td><td>NP00061</td><td></td></tr><tr><td>Amersham Hypond P 0.2 PVDF membrane</td><td>GE Healthcare Life Sciences</td><td>10600021</td><td></td></tr><tr><td>Poly-D-Lysine 8 Well Culture Slide</td><td>Corning BioCoat</td><td>354632</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Normal goat serum (NGS)</td><td>Gibco, Thermo Fisher Scientific</td><td>PCN5000</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L Antibody), Alexa Fluor 488 conjugate</td><td>Thermo Fisher Scientific</td><td>A-11001</td><td></td></tr><tr><td>Goat anti-rabbit IgG (H+L Antibody), Alexa Fluor 568 conjugate</td><td>Thermo Fisher Scientific</td><td>A-11011</td><td></td></tr><tr><td>4&amp;prime;, 6-Diamidin-2-phenylindol (DAPI)</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>Duolink PLA probe Anti-mouse PLUS</td><td>Sigma</td><td>DUO92001</td><td></td></tr><tr><td>Duolink PLA&amp;nbsp; probe Anti-rabbit MINUS</td><td>Sigma</td><td>DUO92005</td><td></td></tr><tr><td>Duolink Detection Reagents Red</td><td>Sigma</td><td>DUO92008</td><td></td></tr><tr><td>Duolink Wash Buffers Fluorescence</td><td>Sigma</td><td>DUO82049</td><td></td></tr><tr><td>Duolink Mounting Medium with DAPI</td><td>Sigma</td><td>DUO82040</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma</td><td>81381</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>AX-70</td><td></td></tr><tr><td>CCD camera</td><td>SPOT</td><td>Insight 2MP Firewire</td><td></td></tr><tr><td>X-ray film</td><td>Fujifilm</td><td>Super RX</td><td></td></tr><tr><td>Film processing machine</td><td>Fujifilm</td><td>FPM-100A</td><td></td></tr></tbody></table>

visualization of protein-protein interaction in nuclear and cytoplasmic fractions by co-immunoprecipitation and in situ proximity ligation assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

도 1 및 NF90 RBM3 모두 핵 단백질과 작은 부분이 세포질에 존재하는 것을 보여준다. 특히, RBM3에 대한 긍정적 인 스테인드 세 가지 밴드가 있습니다. 20 kDa의 아래 작은이 RBM3의 정확한 크기를 반영 (RBM3의 예측 분자량이 17 kDa의)입니다. 다른 두 밴드의 기원은 조사가 남아있다. 미끼 단백질로 RBM3와 공동 면역 침전 실험 NF90-RBM3 상호 작용은 핵 및 세포질에서 소수 ?...

Watch this Scientific Journal Video about Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay at JoVE.com

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

공동 면역 침전에 의한 핵 및 세포질 분수에서 단백질 - 단백질 상호 작용의 시각화 및<em&gt; 현장에서</em&gt; 근접 내고 분석

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

visualization-protein-protein-interaction-nuclear-cytoplasmic

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

12.7K 조회수.  University of Basel. Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

비디오: Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

Proximity Ligation Assay를 이용한 현탁 세포 배양에서의 DNA 손상 유발 단백질 복합체의 검출 및 가시화

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

연구

생화학

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

제자리에 근접 결 찰 분석 결과 사용 하 여 단백질 Isoforms의 Heterodimerization의 검출

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

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 ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

대규모 핵 상호 작용 프로파일링을 위한 라벨 없는 면역 침전 질량 분광 워크플로우

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

근접 결찰 분석술을 사용하여 C. 예쁜꼬마선에서 리보뉴클레오 단백질 복합어셈블리의 시투 검출

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

발생학

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

검출<em&gt; 현장에서</em상기&gt; 단백질 - 단백질 복합체<em&gt; 초파리</em근접 내고 분석을 사용하기&gt; 애벌레 신경 근육 접합부

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

신경과학

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

이미징 단백질 - 단백질 상호 작용 생체내에</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

생물학

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

박테리아, 효모 및 인간 세포에서 일시적으로 형성된 분자 샤페론 어셈블리의 시투 모니터링

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (&gt;40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

근접 결찰 분석에 의한 ER-미토콘드리아 접촉 부위에서 내인성 세포 기관 내 단백질 상호 작용의 시각화 및 정량화

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium ...

<meta charset="utf8"></head><body>DNA 손상에서 단백질 상호 작용 및 국소화 평가

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.

<meta charset="utf8"></head><body>저자 스포트라이트: DNA 손상 반응에서 단백질 상호 작용을 특성화하기 위해 근접 리간드 분석과 Gamma-H2AX 염색의 결합

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

DNA 손상 부위의 단백질을 국소화하기 위한 Proximity Ligand Assay

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that ...

공동 면역 침전에 의한 핵 및 세포질 분수에서 단백질 - 단백질 상호 작용의 시각화 및

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