Name: a protocol for the production of klrg1 tetramer
Uploaded: 2010-01-12T16:01:00
Description: Watch this Scientific Journal Video about A Protocol for the Production of KLRG1 Tetramer at JoVE.com

我们感谢博士Naidenko优化复性条件的帮助。这项工作是支持由美国国立卫生研究院的研究经费AI58181洛朗Brossay。辛迪迪班是由美国国立卫生研究院NRSA F31 AI080230支持。 斯蒂芬妮Terrizzi和辛迪迪班平等作出贡献，对这项工作。

包涵体的制备<ol><li>接种4 × 500毫升烧瓶结核病每个过夜培养的细菌，表达KLRG1质粒构建含有50微克/ ml的羧苄青霉素和氯霉素。当外径达到0.6 A，在600 nm与0.4 M IPTG诱导，并在37孵育一个附加的4小时晃动文化° C。 </li><li>中提取的干细胞重悬在再悬浮缓冲液的pH值= 8.0（50 25％（W / V）蔗糖，1 mM的EDTA，MM的Tris - HCl，10 mM的数码地面电视）。注：颗粒可以在这个阶段被冻结。 </li><li>池不超过60毫升细菌res...

<ol>
	<li>Tessmer, M. S., Fugere, C., Stevenaert, F., Naidenko, O. V., Chong, H. J., Leclercq, G., Brossay, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KLRG1+binds+cadherins+and+preferentially+associates+with+SHIP-1.">KLRG1 binds cadherins and preferentially associates with SHIP-1.</a> Int Immunol. 19, 391-400 (2007).</li><li>Grundemann, C., Bauer, M., Schweier, O., von Oppen, N., Lassing, U., Saudan, P., Becker, K. F., Karp, K., Hanke, T., Bachmann, M. F., Pircher, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+identification+of+E-cadherin+as+a+ligand+for+the+murine+killer+cell+lectin-like+receptor+G1.">Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1.</a> J Immunol. 176, 1311-1315 (2006).</li><li>Ito, M., Maruyama, T., Saito, N., Koganei, S., Yamamoto, K., Matsumoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killer+cell+lectin-like+receptor+G1+binds+three+members+of+the+classical+cadherin+family+to+inhibit+NK+cell+cytotoxicity.">Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK cell cytotoxicity.</a> J Exp Med. 203, 289-295 (2006).</li><li>Li, Y., Hofmann, M., Wang, Q., Teng, L., Chlewicki, L. K., Pircher, H., Mariuzza, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+natural+killer+cell+receptor+KLRG1+bound+to+E-cadherin+reveals+basis+for+MHC-independent+missing+self+recognition.">Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.</a> Immunity. 31, 35-46 (2009).</li><li>Nakamura, S., Kuroki, K., Ohki, I., Sasaki, K., Kajikawa, M., Maruyama, T., Ito, M., Kameda, Y., Ikura, M., Yamamoto, K., Matsumoto, N., Maenaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K.+Molecular+basis+for+E-cadherin+recognition+by+killer+cell+lectin-like+receptor+G1+(KLRG1).">K. Molecular basis for E-cadherin recognition by killer cell lectin-like receptor G1 (KLRG1).</a> J Biol Chem. , .</li></ol>

在这个协议中，它是如何净化，biotinylate，tetramerize KLRG1。 KLRG1四聚体可以用来标记流式细胞仪KLRG1配体。虽然复性条件进行测试，每个蛋白质的经验，其他的C型凝集素可tetramerized使用了类似的协议。使用tetramerized蛋白作为探针，对孤儿的C型凝集素受体的配体可能被确定。

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		<th>Material Name</th>
		<th>Type</th>
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<td>BirA Enzyme and Buffer</td>
<td>Biotinylation Kit</td>
<td>Avidity</td>
<td>BIRA500</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitor Tablets, EDTA Free</td>
<td>Reagent</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>or equivalent</td>
</tr>
<tr>
<td>Amicon Stirred Cell</td>
<td>Equipment</td>
<td>Millipore</td>
<td>Model #8400</td>
<td>or equivalent</td>
</tr>
<tr>
<td>YM10 NMWL 10K ultrafiltration membrane</td>
<td>Filtration Supply</td>
<td>Millipore</td>
<td>13642</td>
<td>&nbsp;</td>
<tr>
<td>15 ml YM10 concentrator</td>
<td>Filtration Supply</td>
<td>Millipore</td>
<td>4411</td>
<td>&nbsp;</td>
<tr>
<td>AKTAFPLC</td>
<td>Equipment</td>
<td>GE Healthcare</td>
<td>18-1900-26</td>
<td>&nbsp;</td>
<tr>
<td>Sephacryl S-200 16/60 high-resolution column</td>
<td>Equipment</td>
<td>GE Healthcare</td>
<td>17-1166-01</td>
<td>&nbsp;</td>
<tr>
<td>Strepavidin PE</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>554061</td>
<td>or equivalent</td>
</tr>		</tbody>
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a protocol for the production of klrg1 tetramer

杀伤细胞凝集素样受体G1（KLRG1）是II型跨膜糖蛋白抑制性受体，属于C型凝集素样超家族。 KLRG1存在单体和二硫键相连的二聚体。这种保守的受体上最成熟，最近激活NK细胞，以及效应/记忆性T细胞的一个子集。 使用KLRG1四聚体，以及其他的方法，E，N，和R -钙粘素被确定为KLRG1配体。这些CA 2 +依赖的细胞，细胞粘附分子由胞外域包含五个cadherin的细胞间的相互作用，跨膜结构域和细胞质域与肌动蛋白细胞骨架的重复。 KLRG1四聚体的生成，是必不可少的KLRG1配体的识别。 KLRG1四聚体，也是一个独特的工具，以澄清在调节免疫反应和组织的完整性的作用cadherin和KLRG1发挥。

Watch this Scientific Journal Video about A Protocol for the Production of KLRG1 Tetramer at JoVE.com

A Protocol for the Production of KLRG1 Tetramer

这个协议描述KLRG1四聚体的生产，这是一个强大的工具KLRG1配体的分析。

KLRG1四聚体的生产协议

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

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