이 작품은 공중 보건의 캘리포니아학과에서 래리 L. Hillblom 알츠하이머 협회 재단 IIRG - 07-58334, 07-65798에서와 NIH / NIA, 2005/2E에서 보조금 AG027818 및 AG030709 지원했다.

1. 펩타이드 준비 <ol><li> 라벨, 실리콘 코팅, 낮은 흡착제 microfuge 튜브에 microbalance 및 전송을 사용하여 동결 건조된 펩타이드의 ~ 100-200 μg를 달다. 여기, 우리는 Aβ40의 인간 시퀀스, Aβ42, 칼시토닌, 그리고 GRF를 사용합니다. </li><li> 여기, 우리는 단일 펩티드, 집계없는 준비를 얻기 위해 1,1,1,3,3,3 - hexafluoro - 2 - 프로파놀 (HFIP)로 사전 처리를 사용합니다. 사전 구성된 집계가 실험 5 중에서 가난한 재현성의 결과 amyloidogenic 단백질의 신속한 집계를 유발하기 때문에이 단계가 필요합니다. 이러한 여과 및 SEC와 같은 다른 방법도 PICUP 6 총체적없는 준비를 얻는 데 사용할 수 있습니다. </li><li> HFIP와 펩티드를 치료하기 위해 적절한 보호 (HFIP는 휘발성과 독성)을 입고 연기 후드 안에 얼음 HFIP 컨테이너를 미리 진정해. 250 ML 병의 냉각하면 일반적으로 10-15 분 필요합니다. 0.5 mm의 공칭 펩타이드 농도를 구하는 펩티드 lyophilizates를 포함하는 미리 차게 튜브에 HFIP를 추가합니다. </li><li> 상온에서 5 분 물 목욕 sonicator의 펩타이드 솔루션을 Sonicate. </li><li> 부드럽게 소용돌이와 실온에서 30 분 튜브를 품어. </li><li> 얼음 (1 분)에 튜브를 칠하고, 레이블 0.6 - ML 낮은 흡착제 튜브에서 10-50 - μl aliquots에 솔루션을 나눕니다. </li><li> 질소의 부드러운 흐름에 따라 증발에 의해 HFIP를 제거하거나, 하룻밤 퓸 후드에서 열 튜브를 두십시오. 하룻밤 증발 들어, 랙에 오픈 튜브를 삽입하고 먼지와 미립자 오염을 방지하기 위해 Kimwipes의 큰 시트를 커버. </li><li> lyophilizer에 vacuo에 남아있는 HFIP, 30 분 원심 농축기를 바싹 말리다, 또는 2 H.위한 진공 입구에 부착된 exsiccator에 최종 제품은 microfuge 튜브의 하단에있는 펩타이드 영화 것입니다. 제대로 exsiccated 경우, 튜브가 -20 또는 -80 ° C.에 연장 기간 (개월)에 대한 완벽한 저장할 수 있습니다 </li></ol> 2. HFIP - 대우 펩티드를 Solubilizing과 사진이 교차 연결 <ol><li> 가교 반응, 교차 연결 및 담금질의 시약을 준비 한 요구에 HFIP - 대우 펩티드를 solubilizing 전에. </li><li> 암모늄 persulfate를 달다 (APS, 미스터 228.2 g / 몰)와 10 MM의 인산 나트륨, 산도 7.4에서 20 MM 솔루션을 준비합니다. 솔루션 분명히 때까지 소용돌이를 사용하여 섞는다. </li><li> 10 MM의 인산 나트륨, 산도 7.4의 트리스 1 MM 솔루션 (2,2 - bipyridyl) dichlororuthenium (II) hexahydrate을 (RuBpy 씨, 748.63 g / 몰) 준비합니다. 소용돌이를 사용하여 혼합하고 완전한 해체를 확인합니다. 알루미늄 호일을 사용하여 빛을에서 튜브를 보호합니다. </li><li> 가교 다음과 SDS - PAGE 분석을 위해, 편리한 담금질 시약은 2 5% β - 메르 캅 토 에탄올이다 × SDS - PAGE 샘플 버퍼. 또는, 1 M dithiothreitol (DTT, 씨 154.5 g / 몰) 탈이온수 또는 적절한 버퍼에 사용할 수 있습니다. </li><li> HFIP - 치료 펩타이드 필름은 희석 NaOH 먼저 해산하고 다음 인산 나트륨 버퍼는 ~ 30 μm의 펩타이드의 농도를 얻을에 추가됩니다. 추가 60 MM NaOH는 NaOH와 물을 각각 10 최종 볼륨의 45 %, 구성 이러한 펩타이드 필름이 들어있는 튜브에 탈이온수 다음. 팁 최대 pipetting 아래로 혼합하고, 물 목욕 sonicator 5 분 sonicate를 사용 microfuge 튜브의 내벽에서 펩타이드 필름을 다쳤어요. </li><li> 45% 20 MM의 인산 나트륨, 산도 7.4, pipetting으로 혼합, 10 분 1만6천g에서 원심 분리기를 추가합니다. 부정적인 컨트롤을위한 담금질 시약과 혼합 uncross 연계 펩티드의 aliquots을 따로 설정합니다. </li><li> 1 s의 카메라 셔터 지연을 조정 높은 조사 시간에 광범위한 급진적인 반응 단백질 저하의 원인이 될 수 있습니다. 카메라 셔터를로드합니다. 조사 시간은 새로운 단백질과 방법을 사용할 때 최적화할 필요가 있습니다. </li><li> 전형적인 PICUP 반응은 20 μl 반응 볼륨에서 수행됩니다. , 얇은 벽으로 둘러싸인, 맑은 0.2 - ML PCR 튜브에 펩타이드 솔루션을 18 μl을 전송합니다. </li><li> 펩타이드 솔루션, 1 μl APS 다음 1 μl RuBpy를 추가하고 (반응 혼합물에 RuBpy와 APS의 최종 농도가 0.05 및 1 ㎜ 각각됩니다) pipetting하여 시약을 섞는다. </li><li> 신속하게 반응 관 1.8 ML의 유리관 안에 넣습니다. 카메라 본체의 전면에 붙어있는 벨로우즈 내부의 병을를 놓습니다. 렌즈 보호를 첨부와 샘플이 벨로우즈 내부 1 S에 대한 조사가되도록 셔터를 누르십시오. </li><li> 샘플 조사 후, 신속하게 유리병 밖으로 풀무와 PCR 튜브의 병을 꺼내. 신속 한 μl DTT, 10 μl 절감 페이지 샘플 버퍼를 추가하여 반응은 끄지. 각 펩타이드 나누어지는에 대한 반응을 반복합니다. </li><li> 반응 혼합물은 이제 노 칠일 이상 또는 SDS - PAGE와 실버 얼룩으로 분석하기 전에 얼음에 보관을위한 스토리지 -20 ° C에 냉동 수 있습니다. </li></ol> 3. SDS - PAGE와 은색 - 염색법교차 연결된 펩타이드 제품 <ol><li> 정기적인 SDS - PAGE와 실버 얼룩이 교차 연결된 펩티드를 시각화하기 위해 수행됩니다. </li><li> 차선 당 펩티드의 ~ 130 pmol를 얻을 레인 당 반응 혼합물의 5 μl를로드합니다. </li><li> 비교 uncross 연계 펩티드 비슷한 금액을 포함합니다. 펩타이드 밴드의 분자량의 시각 근사치에 대한 표준 단백질 사다리도 포함됩니다. </li><li> 표준 겔 전기 영동 장치를 사용하여 겔을 실행합니다. 우리는 Invitrogen에서 XCell SureLock 미니 셀 시스템을 사용하고 Invitrogen 출판 IM - 1002, Novex 사전 캐스트 젤 전기 영동 안내에 따라 실버 얼룩을 수행합니다. </li></ol> 4 부 : 대표 결과 (그림 1) <img src="/files/ftp_upload/old/1071_2009_1_19_13/Result no demarcation.png" alt="그림 1" /> 그림 1 : uncross 링크된 보여주는 실버 - 스테인드 polyacrylamide의 젤 - (+) GRF, 칼시토닌, Aβ40과 Aβ42과 사진 - 상호 연결된 ()를 SDS - PAGE와 PICUP 생성 Aβ40 oligomers의 실버 얼룩 분석은 높은 oligomers 7 풍부하게 날카로운 감소 다음 tetramer를 통해 모노머 약 유사한 밴드 농도를 보여줍니다. Uncross 링크된 Aβ40는 모노머 7 그것과 일치 미스터와 함께 마이 그 레이션합니다. Aβ42는 뚜렷한 올리고머 크기 분포를 보여줍니다. Aβ42 oligomers 2-3 그룹 8 구성됩니다. 첫 번째 그룹, trimer를 통해 모노머가 증가 올리고머 주문과 함께 강도를 감소 표시됩니다. 두 번째 그룹에서, 가우스 같은 배포 pentamer과 hexamer에서 최대, tetramer 그리고 heptamer 사이에 관찰됩니다. 여기에 표시되지 않습니다 세 번째 그룹은, 씨 ~ 30,000-60,000 다 8 oligomers가 포함되어 있습니다. 이러한 높은 미스터 oligomers는 일반적으로 SEC 절연 LMW Aβ42를 사용하여 관찰 있지만 펩티드는 여과 또는 HFIP 치료 6 준비가되어 있습니다. Uncross 링크된 Aβ42은 주로 두 그룹, 모노머의 밴드와 SDS 5,9에 의해 유도된 인위입니다 광범위한 trimer / tetramer 밴드를 생산하고 있습니다. 칼시토닌 강력 dimers, trimers, 그리고 tetramers 7 미리 존재를 제안, tetramer를 통해 지역 모노머의 단조로운 지수 감소에서 diverges 올리고머 크기 분포를 산출. 제어 polypeptide, GRF은 이합체에서 oligomers의 명백한 상대적 금액이 증가 분자 질량과 함께 기하 급수적으로 감소되는 등 hexamer를 통해 범위 monotonic 올리고머 분포를 생산하고 있습니다. 다른 실험에서, 높은 oligomers 또한 시각 7 수 있습니다. 정확한 번호와 올리고머 밴드의 상대적 농도는 실제 단백질 농도, 젤에 로드된 단백질의 양, 그리고 실버 얼룩 프로토콜의 개발 단계에 사용되는 시간에 따라 실험을 사이에 다소 차이가있을 수 있습니다.

<ol>
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PICUP는 안정적인 단백질 단지 2 공부 원래 개발되었습니다. 방법은 Aβ 10, 프리온 질병 - 관련 PrP SC 11, 및 α - synuclein 12를 포함한 metastable 아밀로이드 단백질 어셈블리의 양적 연구 나중에 적용되었다. PICUP 실험을 디자인할 때 고려되어야하는 가장 중요한 요소는 시약의 stoichiometry, 조사 시간 및 샘플 준비 절차입니다. 후자 문제가 크게 실험 데이터의 해석에 영향을 미치는 반면 전...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dolan-Jenner 200-W incandescent lamp</td>
<td>Other</td>
<td>Dolan-Jenner Industries</td>
<td>Model 170-D</td>
<td>The heat generated by the lamp does not affect samples for short incubation periods.</td>
</tr>
<tr>
<td>35-mm SLR camera body</td>
<td>Tool</td>
<td>Pentax</td>
<td>SP500 model</td>
<td>In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.</td>
</tr>
<tr>
<td>Clear, thin walled PCR tubes</td>
<td>Other</td>
<td>Eppendorf</td>
<td>951010006 supplied by Fisher L22-003-24</td>
<td>&nbsp;</td>
<tr>
<td>Glass vials (1.8 mL)</td>
<td>Other</td>
<td>Kimble Chromatography</td>
<td>60940A 2, supplied by Fisher 03-340-60</td>
<td>&nbsp;</td>
<tr>
<td>GRF</td>
<td>Reagent</td>
<td>Bachem</td>
<td>H-3695</td>
<td>&nbsp;</td>
<tr>
<td>HFIP</td>
<td>Reagent</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>A&beta;40 and A&beta;42</td>
<td>Reagent</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calcitonin</td>
<td>Reagent</td>
<td>American Peptide</td>
<td>22-1-10</td>
<td>&nbsp;</td>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. </td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7154-25 ML</td>
<td>Can be used when SDS-PAGE analysis is performed.</td>
</tr>
<tr>
<td>Novex Tricine SDS Sample Buffer (2x)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10-20%)</td>
<td>Other</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10x )</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Silver Express Staining Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6100</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides

독성 oligomers로 amyloidogenic 단백질의 조립은 알츠하이머, 파킨슨병과 헌팅턴의 질병, 유전 루게릭병, 그리고 2 형 당뇨병을 포함한 단백질 misfolding 질환의 pathogenesis에 있게한 이벤트입니다. 이러한 단백질 어셈블리의 metastable 특성 때문에, 그것은 전기, 크로마 토그래피, 형광, 또는 동적 광 산란과 같은 고전적인 방법을 사용하여 양적 자신의 올리고머의 크기 분포를 평가하기 어렵습니다. amyloidogenic 단백질 Oligomers은 oligomers가 단량체로 교제를 끊다 동시에 큰 어셈블리에 연결할하는 metastable 혼합물로 존재합니다. PICUP 교차하기 전에 존재 올리고머 크기 배포판 공유 결합 가교와 같은 나트륨 dodecyl 황산염의 polyacrylamide 젤 전기 영동 (SDS - PAGE) 또는 크기 배제 크로마 토그래피 (SEC)와 같은 분류 방법과 결합하면 PICUP이 제공하는 스냅샷의 올리고머의 인구 안정화 - 연결. 따라서, PICUP는 metastable 단백질 인구의 시각화 및 정량 분석​​을 가능하게하고 시퀀스 수정 및 oligomerization 사이의 조립 및 해독 관계를 모니터링하는 데 사용할 수 있습니다<sup&gt; 1</sup&gt;. Mechanistically, PICUP는 루의 사진 산화를 포함<sup&gt; 2 +</sup&gt; 트리스 (bipyridyl) 루 (II) 복잡한 (RuBpy)에 루에<sup&gt; 3 +</sup&gt; 전자 수용체의 존재에 가시 광선과 방사선에 의해. 루<sup&gt; 3 +</sup&gt; 단백질 급진적인 생성, 이웃 단백질 분자에서 전자를 추출 수있는 강력한 한 전자 산회제입니다<sup&gt; 1,2</sup&gt;. 부수는 불안정, 높은 반응 종이며, 따라서 내부 및 분자간 반응의 다양한 통하여 급속하게 사라집니다. 급진은 이후 수소 원자를 잃고 안정, covalently 링크된 이합체을 형성 dimeric 급진를 형성 다른 단백질 단량체와 반응하는 unpaired 전자의 높은 에너지를 활용할 수 있습니다. 이합체 다음 고차원 oligomers를 형성하는 단량체 또는 다른 dimers와 유사한 메커니즘을 통해 추가로 반응 수 있습니다. 다른 사진이나 화학 가교 방법에 PICUP 상대의 장점<sup&gt; 3,4</sup&gt; 짧은 (≤ 1 S) 비파괴 가시 광선에 대한 노출에 대한 필요가 포함<i&gt; 예약상의</i&gt; 기본 순서의 변경, 그리고 길이가 0 인 가교 공유 결합. 또한, PICUP는 수 간 연결 넓은 산도와 physiologic 매개 변수를 포함하여 온도 범위 내에서 단백질의. 여기, 우리는 세 amyloidogenic 단백질 40의 상호 연결에 PICUP의 응용 프로그램을 보여 - 42 - 잔류 아밀로이드 β 단백질의 변종 (Aβ40과 Aβ42) 및 칼시토닌 및 제어 단백질, 성장 호르몬 방출 인자 (GRF).

Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

사진 유발 가교 수정되지 않은 단백질 (PICUP)의 것은​​ metastable 단백질 혼합물의 올리고머 크기 분포의 특성을 허용합니다. 우리는 세 대표 amyloidogenic 펩티드 40 PICUP의 응용 프로그램을 보여줍니다 - 그리고 아밀로이드 β 단백질 및 칼시토닌의 42 잔여물 형태 및 제어 펩타이드 성장 호르몬 방출 인자.

사진 유발 가교 수정되지 않은 단백질 (PICUP) Amyloidogenic 펩티드에 적용

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

photo-induced-cross-linking-unmodified-proteins-picup-applied-to

Research

JoVE Journal

Biology

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

21.2K 조회수.  University of California, Los Angeles. 사진 유발 가교 수정되지 않은 단백질 (PICUP)의 것은​​ metastable 단백질 혼합물의 올리고머 크기 분포의 특성을 허용합니다. 우리는 세 대표 amyloidogenic 펩티드 40 PICUP의 응용 프로그램을 보여줍니다 - 그리고 아밀로이드 β 단백질 및 칼시토닌의 42 잔여물 형태 및 제어 펩타이드 성장 호르몬 방출 인자.

비디오: Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

단백질의 전기 영동 분리

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

연구

생물학

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

젤의 단백질 염색법

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

복잡한 생물 학적 샘플에서 선택적으로 - glycosylated 펩티드을 풍요롭게하는 렉틴 HPLC 방법

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

불안 정한 다중 단백질 단지의 분리에 의해 생체내에 제어 세포 간 연결 및 단백질 자기 동질 크로마 토그래피

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

레이저 Microdissection는 세포의 하위 집합 유전자 발현 프로 파일링에 적용 Drosophila 윙 디스크

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

에서 생산 재조합 이황화 풍부한 독 단백질에 적용되는 높은 처리량 정량 식 검사 및 정화<em&gt; E. 대장균</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

에서 펩타이드 및 단백질과 재조합 엘라스틴 같은 폴리 펩타이드 및 그 융해의 비 크로마토 그래피 정제<em&gt; 대장균</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

MALDI-TOF 질량 분석 및 사용자 지정 데이터베이스의 사용은 고유 동굴 환경 (Kartchner 동굴, AZ, USA)에 토착 박테리아의 특성을

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

색채 금 나노 입자 분석에 적용 구조 전환 앱 타머를 선택하는 방법

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

질량 분석기 분석을위한 펩티드로 조직과 소화에서 세포 외 기질 단백질의 농축

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...