이 연구는 교내 연구, 국립 심장, 폐 및 혈액 연구소, 건강의 국립 연구소의 부서에 의해 지원되었다. S.H.는 일본 공립-민간 파트너십 학생 유학 유학 프로그램의 지원을 받았으며, H.N.과 K.Y.는 용맹및 V.Drug 해외 연수 장학금을 받았습니다.

1. SDS 페이지 및 PVDF 멤브레인으로 전송
<ol><li>SDS-PAGE를 수행하여 상대적인 크기에 따라 단백질을 분리합니다. 참고: 상업용 또는 수제 젤 시스템은 괜찮습니다. 제조업체의 프로토콜을 따르십시오.</li>
	<li>분리된 단백질을 젤에서 PVDF 멤브레인으로 전달합니다. 참고: 일반적인 전송 방법(반건조 또는 습식 전송)이 적합합니다. 제조업체의 프로토콜을 따르십시오.</li>
</ol>2. PVDF 멤브레인 차단
<ol><li>실온에서 교반하여 1h에 적합한 차단 버퍼로 멤브레인을 배양합니다. 참고: 1차 항체 및 검출 시스템에 따라 적절한 차단 버퍼를 선택해야 합니다. 예를 들어, 가장 일반적인 차단제는 BSA, 무지방 건조 우유, 카제인 및 상업용 합성 폴리머입니다. PBS 및/또는 0.1% Tween20을 사용하는 TBS는 가장 일반적으로 사용되는 버퍼입니다. 이러한 차단 단계는 4°C에서 하룻밤 사이에 수행될 수 있다.</li>
</ol>3. 1 차 항체와 인큐베이션
<ol><li>증류수로 10% 면역반응 강화 제-1 용액(IRE-1)을 준비한다. 소용돌이 잘.</li>
	<li>희석된 1차 항체를 10% IRE-1로 준비한다. 부드럽게 충분히 섞으세요.</li>
	<li>더 큰 멤브레인(4cm x 8cm - 8cm x 8cm)을 사용하는 경우, 항체 용액8mL을 50mL 원심분리관에 넣습니다. 더 작은 멤브레인(2cm x 8cm - 4cm x 8cm)을 사용하는 경우, 항체 용액3mL을 14mL 원형 바닥 폴리프로필렌튜브(재료 표)에넣습니다.</li>
	<li>핀셋으로 PVDF 멤브레인을 집어 들고 블로킹 용액을 간단히 배출하십시오. 장갑을 낀 손가락으로 PVDF 멤브레인을 이 튜브에 삽입하고 전체 멤브레인이 튜브 벽에 부착되도록 합니다. PVDF 멤브레인이 튜브에 삽입되면 원래 안쪽에 겔과 접촉했던 멤브레인의 "단백질 측면"에 직면하십시오. 캡을 단단히 닫습니다.</li>
	<li>50mL 튜브를 유리 병에 삽입하여 혼성화 오븐에 넣습니다. 참고: 이 배양에 14mL 튜브를 사용하면 플라스틱 링 홀더 쌍을 사용하여 50mL 튜브의 중앙에 내부 튜브를 유지하여 50mL 튜브에 14mL 튜브를 삽입하십시오.</li>
	<li>혼성화 오븐을 켭니다.</li>
	<li>6rpm 회전으로 멤브레인을 5분 이상 배양합니다. 참고: 멤브레인이 항상 벽에 부착되어 있고 (내부) 튜브가 수평으로 수평되어 항체 용액으로 멤브레인을 균일하게 덮는지 확인하십시오. 실제 실험 전에 항체 및 배양 시간의 희석을 교정한다.</li>
</ol>4. 멤브레인 워시
<ol><li>회전을 중지합니다.</li>
	<li>핀셋으로 튜브에서 PVDF 멤브레인을 조심스럽게 제거하고 멤브레인을 PBS-T50mL의 용기에 넣습니다.</li>
	<li>PBS-T로 멤브레인을 간단히 헹구는 다.</li>
	<li>거품이 나타나지 때까지 증류수로 용기에 멤브레인을 헹구는 다. 이것은 항체의 대부분을 제거하는 것입니다.</li>
	<li>PVDF 멤브레인을 용기에서 PBS-T 250mL가 들어 있는 샐러드 스피너로 옮기습니다.</li>
	<li>스피너에 스트레이너 바구니를 놓습니다. 뚜껑을 단단히 고정했는지 확인합니다.</li>
	<li>스피너를 활성화하고 약 20-30 s로 실행합니다.</li>
	<li>용액을 버리고 스피너 내부를 증류수로 간략하게 헹구십시오.</li>
	<li>다시 스피너에 PBS-T의 250 mL을 추가합니다.</li>
	<li>4.6 및 4.7 단계를 반복합니다.</li>
</ol>5. 이차 항체를 가진 배양
<ol><li>증류수로 10% 면역반응 강화 제2용액(IRE-2)을 준비한다. 소용돌이 잘.</li>
	<li>희석된 이차 항체를 10% IRE-2로 준비한다. 부드럽게 충분히 섞으세요.</li>
	<li>3.3 단계에서 명시된 바와 같이 항체 용액 및 튜브의 부피를 선택한다.</li>
	<li>핀셋으로 PVDF 멤브레인을 집어 들고 용액을 간단히 배출하십시오. 3.4 단계에서 명시된 대로 PVDF 멤브레인을 튜브에 삽입합니다.</li>
	<li>50mL 튜브를 유리 병에 삽입하여 혼성화 오븐에 넣습니다.</li>
	<li>혼성화 오븐을 켭니다.</li>
	<li>6rpm 회전으로 멤브레인을 5분 이상 배양합니다. 멤브레인이 항상 벽에 부착되어 있는지 확인하고 (내부) 튜브가 항체 용액으로 멤브레인을 균일하게 덮기 위해 수평인지 확인하십시오. 참고: 실제 실험 전에 인큐베이션 시간을 보정합니다. 이차 항체가 형광으로 수렴되면 어둠 속에서 배양을 수행합니다.</li>
</ol>6. 멤브레인 워시
<ol><li>4.1~ 4.10단계를 따르십시오.</li>
</ol>7. 이미지 수집
<ol><li>화학 발광 검출<ol>
<li>평평한 표면에 반 이식 플렉시블 필름(10cm x 15cm)을 놓습니다.</li>
		<li>5mL 튜브에 화학 발광 기판의 2 성분을 혼합합니다.</li>
		<li>혼합 기판의 1.5mL를 반이식 플렉시블 필름으로 즉시 이송하고 PVDF 멤브레인을 배치한다. 그런 다음 기판 용액의 나머지 부분을 멤브레인으로 이송합니다.</li>
		<li>PVDF 멤브레인을 반투명 플렉시블 필름에 혼합 기판으로 1분 동안 배양합니다.</li>
		<li>핀셋으로 PVDF 멤브레인을 선택하고 기판 용액을 간단히 배출하십시오.</li>
		<li>투명 필름 한 쌍 사이에 멤브레인을 샌드위치.</li>
		<li>화학 발광 모드에서 이미지를 획득합니다.</li>
	</ol>
</li>
	<li>형광 검출<ol>
<li>핀셋으로 PVDF 멤브레인을 집어 들고 용액을 간단히 배출하십시오.</li>
		<li>투명 필름 한 쌍 사이에 멤브레인을 샌드위치.</li>
		<li>형광 모드에서 이미지를 획득합니다. 참고: 7.1 단계와 7.2 단계는 최대 감도를 보장하기 위해 이미징 기계와 가까운 곳에서 수행해야합니다.</li>
		<li>배경 신호가 높을 때, PBS-T의 80-100 mL를 가진 용기에서 세척 단계를 수행: 1차 항체에 대해 10 분 헹구고 이차 항체에 대해 6 배 헹구는 다. 참고: 10% IRE 용액으로 희석된 1차 및 이차 항체 는 감도6의손실 없이 최대 8-10회까지 사용할 수 있다. 용액은 최대 1 개월 동안 4 °C로 유지해야합니다.</li>
	</ol>
</li>
</ol>

<ol>
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저자는 이 문서의 내용과 직접적인 관련이 있는 경쟁적 이해관계를 선언하지 않았습니다.

웨스턴 블롯은 40년 전 ~40년 전에 개발된 특정 단백질을 검출하기 위해 널리 사용되는 분석 기법으로7,8. 그이후로, 기술은 기술2,9,10,11,12,13의감도, 속도 및 양을 향상시키는 후속 혁신과 함께 진화를 계속...

서양 얼룩 (또한 면역 blot로 알려진) 과학 및 임상 분야의 광범위 에 걸쳐 강력하고 기본적인 기술이다. 이 기술은 단백질1의존재, 상대적 풍부, 상대 분자 질량 및 번역 후 수정을 검사하는 데 사용됩니다. 디지털 이미지 분석과 결합하여, 이 방법은 단백질과 단백질 변형의 풍요로움을 안정적으로 분석할 수있다 2. 서양 얼룩은 일상적으로 수행되지만 시간이 많이 걸리고 노동 집약적인 방법입니다. 막이 있는 항체의 긴 배큐어가 필요합니다. 여기서는 민감도를 희생하지 않고 이러한 제한을 극복하는 인큐베이션 방법의 수정을 설명합니다.
막의 인큐베이션 도중, 항원은 막에 고정되는 동안 항체는 용액에 떠 있습니다. 그들의 높은 친화성 때문에, 항원에 결합하는 항체의 비율은 막에 벌크 용액에서 항체의 확산 보다는 빠릅니다. 이렇게 하면 농도가 낮은 "고갈 층"(그림1)이생성됩니다. 이 기술의 긴 잠복 시간을 담당하는 주요 인자인 수동 확산을 통해 더 먼 항체가 막에 도달하는 데 몇 시간이 걸릴 수 있습니다. 이 효과를 MTL(대량 전송 제한)3이라고합니다. 항체 함유 용액을 반복적으로 배수하고 보충하면 고갈층을 방해하고 MTL4를극복할 수 있다는 것이 제안되었다. 여기서, 우리는 전통적인 서쪽 blotting 프로토콜에서 MTL의 효과를 감소시키고 현저하게 필요한 잠복기를 단축하기 위해이 순환 배수 및 보충 (CDR) 개념을 구현하는 독특한 기술을 고안했다.
짧은 배양 시간으로 검출 감도를 유지하기 위해 항원-항체 반응을 가속화하는 면역 반응 강화 기술을 사용하여 신호 대 잡음 비5를개선하였다. 많은 시판 가능한 면역 반응 향상 제 (IRE)는 2 개의 구성 요소 (솔루션 1 및 2, 재료 표참조)로 구성됩니다. IRE의 독점적 조성 또는 작용 메커니즘은 공개되지 않았지만, 우리는 이전에 IRE가 고체 상 결합 작용에서 항원과 항체 사이의 해리상수를 감소시켰으며, 증가친화도를 나타내는 것은 적어도 부분적으로 IRE6의강화 효과에 책임이 있음을 발견하였다. CDR과 IRE의 조합은 감도를 희생하지 않고 전체 프로시저 시간을 줄이는 초고속 서부 블로팅 프로토콜을 생성합니다.

<table>
	<tbody>
		<tr>
			<td>14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap</td>
			<td>Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Apollo Transparency Film for Laser Printers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any kinds of Laser Printer Transparency Film are fine.</td>
		</tr>
		<tr>
			<td>Azure Imaging System 600</td>
			<td>Azure Biosystems</td>
			<td>Azure 600</td>
			<td>Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.</td>
		</tr>
		<tr>
			<td>Can Get Signal Immunoreaction Enhancer Solution</td>
			<td>TOYOBO</td>
			<td>NKB-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CARNATION Instant Nonfat Dry Milk</td>
			<td>Carnation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-mouse IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from sheep</td>
			<td>GE Healthcare</td>
			<td>NA9310V</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from donkey</td>
			<td>GE Healthcare</td>
			<td>NA9340V</td>
			<td></td>
		</tr>
		<tr>
			<td>HYBAID Micro-4</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.</td>
		</tr>
		<tr>
			<td>Immobilon-P PVDF Membrane, 0.45 &#181;m pore size</td>
			<td>Millipore Sigma</td>
			<td>IPVH304F0</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-32211</td>
			<td></td>
		</tr>
		<tr>
			<td>KPL LumiGLO Reserve Chemiluminescent Substrate</td>
			<td>seracare</td>
			<td>5430-0049</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#223; actin antibody</td>
			<td>Millipore Sigma</td>
			<td>A5316</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer (PBS)</td>
			<td>LICOR</td>
			<td>927-40100</td>
			<td>Blocking Buffer for fluorescent detection</td>
		</tr>
		<tr>
			<td>OXO Salad Spinner</td>
			<td>OXO</td>
			<td>32480V2B</td>
			<td>Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.</td>
		</tr>
		<tr>
			<td>Parafilm Sealing Film</td>
			<td>Bemis</td>
			<td>Parafilm M PM996</td>
			<td>semi-transparent flexible film</td>
		</tr>
		<tr>
			<td>Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Rings to hold 14 ml tube in the center of 50 ml tube</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Prepared in a machine shop</td>
		</tr>
		<tr>
			<td>Rabbit anti-6X His tag antibody</td>
			<td>Abcam</td>
			<td>ab9108</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Millipore Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultra-high-speed western blot using immunoreaction enhancing technology

서양 블롯 (또한 면역 blot로 알려진) 생물 의학 연구를 위한 정식 방법. 그것은 일반적으로 특정 단백질의 상대적인 크기와 풍부 뿐만 아니라 번역 후 단백질 수정을 결정 하는 데 사용 됩니다. 이 기술은 풍부한 역사를 가지고 있으며 단순성으로 인해 널리 사용됩니다. 그러나, 서쪽 블로팅 절차는 유명한 처리량을 제한하는 긴 잠복기인 중요한 병목 현상으로 완료하는 데 몇 시간, 심지어 며칠이 걸립니다. 이러한 배양 단계는 막에 고정된 항원에 대한 벌크 용액에서 항체의 느린 확산으로 인해 요구된다: 막 근처의 항체 농도는 벌크 농도보다 훨씬 낮다. 여기서, 우리는 항체 용액의 순환 배수 및 보충 (CDR)을 통해 항원 결합을 개선하여 이러한 배양 간격을 극적으로 감소시키는 혁신을 제시한다. 우리는 또한 분석의 감도를 보존하기 위해 면역 반응 향상 기술을 활용했습니다. CDR 방법과 상용 면역반응 강화제의 조합은 출력 신호를 증폭시키고 항체 인큐베이션 시간을 실질적으로 감소시켰다. 그 결과 초고속 서부 얼룩은 감도 손실 없이 20분 만에 달성할 수 있습니다. 이 방법은 화학 발광 및 형광 검출을 모두 사용하여 서부 얼룩에 적용 될 수있다. 이 간단한 프로토콜은 연구원이 많은 견본에서 단백질 발현의 분석을 더 잘 탐구하는 것을 허용합니다.

이 예는 서쪽 블롯에 면역 강화 기술과 함께 CDR 방법의 효과를 보여줍니다. 정적 배큐베이는 PVDF 멤브레인이 항체 용액으로 배양된 반투명 플렉시블 필름에서 수행되었으며, CDR 인큐베이션은 막과 항체 용액(Movie)을 포함하는 튜브를 회전시켜 혼성화 오븐에 있었다. 도 2는 서쪽 얼룩에 화학 발광 검출에 필요한 각각 항원 및 항체의 양을 보였다. 정적 및 CDR 인큐베이션 모두?...

Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

초고속 서양 블로팅 기술은 면역 반응 강화 제와 함께 순환 배수 및 보충 (CDR) 기술을 통해 결합하는 항원 항체의 운동성을 개선하여 개발됩니다.

면역 반응 강화 기술을 이용한 초고속 웨스턴 블롯

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

ultra-high-speed-western-blot-using-immunoreaction-enhancing

Research

JoVE Journal

Biochemistry

5.8K 조회수.  National Institutes of Health. 초고속 서양 블로팅 기술은 면역 반응 강화 제와 함께 순환 배수 및 보충 (CDR) 기술을 통해 결합하는 항원 항체의 운동성을 개선하여 개발됩니다.

비디오: Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

비계 리포좀을 사용하여 근위 지질 단백질 - 단백질 상호 작용을 재구성<em&gt; 체외</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

연구

생화학

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

조 혈 줄기 세포의 작은 숫자에 대 한 프로토콜을 더럽혀 서 부

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

높은 처리량, 양적 도트를 사용 하 여 조직 수준에서 선택 된 단백질의 내용의 절대 결정 오 점 분석 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

단백질 수준 및 개발 표준화 양적 부 럽을 사용 하 여 전체 조직에 걸쳐 강력한 비교

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

이노시톨 인산염 또는 인산성교단백질의 동정은 서부 블롯 또는 질량분광법에 결합된 친화성 크로마토그래피에 의한 단백질

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

식물 MicroRNAs의 검출 및 정량화를 위한 RNA 블롯 분석

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

필드 이펙트 바이오센싱 기술을 이용한 분자 샤페론 Hsp90과 클라이언트 단백질 키나제 Cdc37 사이의 생체분자 상호작용 탐구

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.

The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

필 블롯 기법: 다층 또는 농축된 생물학적 시료에서 단층을 분리하는 Cryo-EM 시료 전처리 방법

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

웨스턴 블로팅을 이용한 뉴런 K-Cl 공동수송체 KCC2의 기능 및 활성 연구

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...