1. 솔루션 및 자료 준비 이러한 단계는 사전에 몇 일 실시하여야한다. <ol><li> 유리 coverslips. 지우지 감동과 함께 3 비율 및 온수 : 물, 1 혼합 : Coverslips는 Linbro 7X 세제의 솔루션으로 10 분 동안 침지하여 세척했다. Coverslips는 탈이온수와 함께 광범위하게 씻어서 후 6 시간 동안 450 ° C에서 구워되었습니다. 세라믹 얼룩 랙에에 coverslips을로드하면 (시약 참조)이 과정을 단순화. </li><li> 스탬프에 대한 단백질 솔루션입니다. 1 MG / ML 농도의 주식 솔루션 제조 업체의 지침에 따라 Reconstitute의 fibronectin. </li></ol> 2. topological 마스터에서 우표를 캐스트 이러한 단계는 사전에 몇 일 실시 수 있습니다. 이러한 조직 문화 요리로 덮여 접시에 저장 우표 패턴 업. <ol><li> 압축, 여과 공기 또는 불활성 가스의 흐름을 사용하여 마스터에서 느슨한 먼지를 제거합니다. </li><li> 주인보다 단지 큰 플라스틱 접시의 바닥에, 마스터, 패턴 측면을 놓으십시오. 60 - 또는 100 - mm 조직 문화 요리가 잘 이러한 목적에 적합합니다. </li><li> 폴리스티렌 50 ML의 원심 관에서 치료 요원의 비율로 Sylgard 구성 요소를 결합 : 1 엘라스토머 기지 : 10 무게로. 같은 일회용 피펫으로 플라스틱 장치를 사용하여 철저히 섞는다. 요리 지역의 cm2 당 탄성체 최소한 0.2 ML를 준비합니다. </li><li> 공기 방울을 제거하는 5 분 300 G에서 50 ML 튜브를 원심 분리기. </li><li> 주인 위로 탄성체를 부어 후 30 분, 진공에서 건조기에서 개최. </li><li> 65 ° C 이상에서 2 시간 동안 오븐에 탄성 치료. 높은 온도에서와 stiffer 탄성체에 더 이상 시간이 결과에 대한 치료. 실온으로 냉각하자. </li><li> 마스터에서 우표의 시트를 분리합니다. </li></ol> 3. 유리에 fibronectin의 Microcontact 인쇄 <ol><li> 하나의 스탬프를 잘라 버릴거야. 4mm을 측정하는 우표는 X 4mm 1cm로 X 1 지역 cm 및 1~2mm 두께로 시작하는 가장 쉬운 방법입니다. 유리 슬라이드 또는 작은 플라스틱 접시에 장소 패턴 측면까지. </li><li> 30 초 동안, 진공에서 플라즈마 청소기, 그리고 과정에서 스탬프를 배치합니다. 가장 높은 출력 설정에서 설정 Harrick 과학 플라즈마 청소기 (장비 참조), PDMS 표면이 친수성 ​​렌더링됩니다. 탄성체의 균열에서 긴 시간 결과. </li><li> 탈이온수와 50 μg / ML의 스탬핑 농도에 fibronectin 솔루션을 희석. </li><li> 우표에 대한 솔루션을 스탬프의 - 작은 방울 (50 μl 10) 장소. 이것은 친수성 ​​표면에 걸쳐 분산됩니다. 드롭이 우표를 다루고 있지만, 가장자리를 통해 실행되지 않는 유일한 충분한 솔루션을 추가합니다. 5 분 스탬프로 단백질 adsorb하자. </li><li> Kimwipe의 ® 또는 기타 깨끗한 종이 티슈를 사용하여 패턴 영역을 건드리지 않고, 우표에서 단백질 솔루션의 대부분을 날려 심지. </li><li> 같은 질소로 깨끗하고, 건조, 불활성 가스의 흐름에 따라 우표의 나머지 솔루션을 건조. </li><li> 족집게를 사용하면, 유리 슬라이드에서 우표를 제거 반전하고, 청소 유리 coverslip (표면 패턴 예정)와 접촉 곳입니다. 좋은 접촉을 촉진하기 위해 상단에 무게를 넣으십시오. 최고의 patterning을 제공합니다 구체적인 무게는 우표 크기와 패턴에 따라 달라집니다, 5g의 무게로 시작하고, stampings 간의 조정합니다. 일분에 대한 표면과 접촉 스탬프 둡니다. </li><li> 조심스럽게 스택을 분해하고, coverslip에서 별도의 도장. </li><li> 적극적으로 표면에 adsorbed되지 않습니다 단백질을 제거하는 탈이온수 다음 PBS의 패턴 coverslip을, 린스. 질소 기류 하에서 건조 coverslip. </li></ol> 대표 결과 Microcontact 인쇄 표면에 patterning 분자위한 강력한 프로세스입니다. 그림,이 과정은 micrometers 수십에서 수백 나노미터에 이르기까지 다양한 크기와 기능을 만들 수있는 능력이 있습니다. 녹색 반점이 그림 삽화가 동안 1A, 왼쪽에있는 로고, 높이 각 200 μm의 수 있습니다. 1B는 직경 1 μm의 있으며 가운데에 중심을 측정 4 μm의 간격에서 간격. 그림. 1B 또한 강력한 그것입니다 즉 microcontact 인쇄의 속성 및 첨가제 과정을 보여줍니다. microcontact 인쇄 여러 라운드는 복수 시스템 3 만드는 하나의 표면에 적용할 수 있습니다. <img src="/files/ftp_upload/old/1065_2008_11_24_3/Figure_a.jpg" alt="그림" /><img src="/files/ftp_upload/old/1065_2008_11_24_4/FIGURE_B.jpg" alt="그림 B" />

<ol>
	<li>Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+control+of+cell+life+and+death.">Geometric control of cell life and death.</a> Science. 276, 1425-1425 (1997).</li><li>Kumar, A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+Gold+Having+Micrometer+to+Centimeter+Dimensions+can+be+Formed+Through+a+Combination+of+Stamping+with+an+Elastomeric+Stamp+and+an+Alkanethiol+"Ink"+Followed+by+Chemical+Etching.">Features of Gold Having Micrometer to Centimeter Dimensions can be Formed Through a Combination of Stamping with an Elastomeric Stamp and an Alkanethiol "Ink" Followed by Chemical Etching.</a> Applied Physics Letters. 63, 4-4 (1993).</li><li>St. John, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+Glial+Cell+Attachment+to+Microcontact-printed+Surfaces.">Preferential Glial Cell Attachment to Microcontact-printed Surfaces.</a> Journal of Neuroscience Methods. 75, 171-171 (1997).</li><li>Kam, L., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+to+protein-micropatterned-supported+lipid+bilayer+membranes.">Cell adhesion to protein-micropatterned-supported lipid bilayer membranes.</a> Journal of Biomedical Materials Research. 55, 487-487 (2001).</li><li>Kung, L. A., Kam, L., Hovis, J. S., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Hybrid+Surfaces+of+Proteins+and+Supported+Lipid+Bilayers.">Patterning Hybrid Surfaces of Proteins and Supported Lipid Bilayers.</a> Langmuir. 16, 6773-6773 (2000).</li><li>Shen, K., Thomas, V. K., Dustin, M. L., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+of+costimulatory+ligands+enhances+CD4++T+cell+function.">Micropatterning of costimulatory ligands enhances CD4+ T cell function.</a> Proceedings of the National Academy of Sciences. 105, 7791-7791 (2008).</li><li>Shi, P., Shen, K., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+presentation+of+L1+and+N-cadherin+in+multicomponent,+microscale+patterns+differentially+direct+neuron+function+in+vitro.">Local presentation of L1 and N-cadherin in multicomponent, microscale patterns differentially direct neuron function in vitro.</a> Developmental Neurobiology. 67, 1765-1765 (2007).</li></ol>

The authors have nothing to disclose.

microcontact 인쇄 과정은 patterning 기판의 다양한 분자의 광범위한 적용을 받으면, 개념 간단하고 매우 견고합니다. 그러나,이 과정은 예술의 무언가 남아있다. 만들 수있는 패턴의 특정 기하학이 단백질 패턴으로 적용, 무게, 도장 / 스탬프 조건은 모든 스탬핑 품질에 영향을 미칩니다. 예를 들어, 자주 큰 기능 그림의 오른쪽 상단 로고에서 볼 수 있듯이 패턴의 격차 결과에 적용도 약간의 무게. 1A. 반대로, 너무 많?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Plasma Cleaner</td>
<td>&nbsp;</td>
<td>Harrick Scientific Products, Inc.</td>
<td>PDC-32G</td>
<td>&nbsp;</td>
<tr>
<td>Desiccator</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5315-0150</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10010-072</td>
<td>&nbsp;</td>
<tr>
<td>Protein labeling kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>A30006</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin </td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F2006</td>
<td>&nbsp;</td>
<tr>
<td>Staining rack</td>
<td>Reagent</td>
<td>Thomas Scientific</td>
<td>8542E40</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>12-544-12</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>Reagent</td>
<td>Ellsworth Adhesives</td>
<td>184 Sil Elast Kit</td>
<td>&nbsp;</td>
<tr>
<td>Diffraction Grating</td>
<td>Reagent</td>
<td>Edmund Scientific</td>
<td>3040267</td>
<td>&nbsp;</td>		</tbody>
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microcontact printing of proteins for cell biology

패턴 단백질과 기판에 다른 biomolecules 수있는 능력은 세포외 환경의 공간적 복잡도를 캡처하는 것이 중요합니다. 와이트 그룹에 의해 microcontact 인쇄의 개발 (<a href="http://gmwgroup.harvard.edu/"&gt; http://gmwgroup.harvard.edu/</a&gt;) 1990 년대 중반 마이크로 일렉 트로닉스 / 생명 과학에 초점을 실험실에 액세스할 수 microfabrication 기술을 제작하여이 필드를 revolutionalized. polydimethylsiloxane (PDMS) 우표는 재료 표면에 작용 화학 물질의 패턴을 만드는 데 사용되는이 방법의 초기 구현<sup&gt; 1</sup&gt;. 이후, 혁신적인 접근 방법의 범위는 단백질을 포함한, 패턴 다른 분자로 개발되었습니다<sup&gt; 2</sup&gt;. 이 동영상은 PDMS 스탬프를 만드는 기본적인 과정을 보여주는 이러한 단계를 정확하게 말로 표현하기가 어렵습니다 같은 패턴 단백질로 사용합니다. 우리는 patterning patterning의 구체적인 예를 들어 유리 coverslips에 세포외 기질 단백질 fibronectin에 중점을두고 있습니다. microcontact 인쇄 과정의 중요한 구성 요소는 우표가 캐스팅되는에서 topological 마스터이며 주인의 인상과 인하 지역은 스탬프로 미러링하여 최​​종 패턴을 정의하고 있습니다. 일반적으로, 마스터가 포토 레지스트로 코팅하고 석판술에 의해 패턴 실리콘 웨이퍼로 이루어져, 여기는 다이기 때문입니다. 특정 패턴을 포함하는 주인의 창조는 전문 장비를 필요로하고, 최고의 제조 센터 또는 시설과 상담에 접근합니다. 그러나, 토폴로지와 함께 거의 모든 기판 등 플라스틱 회절 통같이로 마스터, (한 예를 들어 시약 참조)로 사용하고, 같은 serendipitous 주인은 즉시 사용할 수, 간단한 패턴을 제공할 수 있습니다. 이 프로토콜은 손에 마스터를 둔 지점에서 시작됩니다.

Watch this Scientific Journal Video about Microcontact Printing of Proteins for Cell Biology at JoVE.com

Microcontact Printing of Proteins for Cell Biology

Microcontact 인쇄는 소재 표면에 패턴 단백질 및 다른 분자로 광범위하게 사용됩니다. 우리는 유리에 fibronectin의 패턴을 스탬핑,이 과정의 기본 단계를 보여줍니다.

세포 생물학을위한 단백질의 Microcontact 인쇄

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

microcontact-printing-of-proteins-for-cell-biology

Research

JoVE Journal

Biology

19.9K 조회수.  Columbia University. Microcontact 인쇄는 소재 표면에 패턴 단백질 및 다른 분자로 광범위하게 사용됩니다. 우리는 유리에 fibronectin의 패턴을 스탬핑,이 과정의 기본 단계를 보여줍니다.

비디오: Microcontact Printing of Proteins for Cell Biology

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

세포 생물학을위한 그라디언트 생성 Microfluidic 장치

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

연구

생물학

Micro-scale Engineering for Cell Biology

세포 생물학을위한 미세 규모 공학

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

CyDye DIGE 플루어 최소 염료를 사용하여 셀 표면 단백질의 선택적 Labelling

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

전자 Crystallography에 의해 구조 생물학 연구에 대한 작은 막 단백질의 2 차원 결정화 평가판을 평가

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

COS - 7 형광 라벨링는 라이브 셀 이미징에 대한 SNAP - 태그 퓨전 단백질 표현

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

단백질 막 중첩 분석 : 가용성식이 및 불용성 단백질 간의 상호 작용을 테스트하는 프로토콜 체외에서</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

풀다운 Calmodulin 결합 단백질의

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

성장 팩터 Progranulin와 상호 작용하는 단백질을 식별하기 위해 수정된 효모 - 2 하이브리드 시스템

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Zebrafish 개발하는 동안 Photoconvertible 단백질을 사용하여 셀 추적

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

미생물 시스템 생물학의 Chemostats의 사용

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...