Name: study of the functions and activities of neuronal k-cl co-transporter kcc2 using western blotting
Uploaded: 2022-12-09T13:15:00
Description: Watch this Scientific Journal Video about Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting at JoVE.com

这项工作得到了英国皇家学会（Grant no. IEC\NSFC\201094）和英联邦博士奖学金的支持。

注意：该方案描述了用于检测特定目标蛋白质的蛋白质印迹方法。
1. 细胞培养和转染
<ol><li>在细胞培养过程之前加热珠浴（37°C）中的所有试剂。准备培养基，Dulbecco的改良鹰培养基（DMEM），补充有10%胎牛血清，1%的2mM L-谷氨酰胺，100x非必需氨基酸，100mM丙酮酸钠和100单位/ mL青霉素链霉素。</li>
	<li>解冻稳定转染的大鼠KCC2b人胚胎肾293细胞（HEK293rnKCC2b...

许多方法已被用于测量在神经元中表达的CCC的SLC12的活性，包括KCC2。其中许多技术已被证明可以增强分析这些转运蛋白的功能相关性及其在不同疾病相关突变中的结构 - 功能模式的科学知识。至关重要的是，各种方法都有优点和注意事项21.然而，上面解释的方案概述了如何使用蛋白质印迹评估激酶调控位点Thr906和Thr1007的KCC2磷酸化，这有助于研究KCC2的功能和活性。
<p class="jove...

氯化钾共转运蛋白2（KCC2）是阳离子氯化共转运蛋白（CCCs）溶质载体家族12（SLC12）的成员，仅在神经元中发现，对于Cl-稳态的正常功能以及因此功能性GABA能抑制1，2，3，4至关重要。KCC2将低神经元内Cl-浓度（[Cl-]i）维持在4-6mM，有助于大脑和脊髓中的γ-氨基丁酸（GABA）/甘氨酸超极化和突触抑制5。KCC2的适当调节失败与几种神经系统疾病的流行有关，包括癫痫4。此外，KCC2介导的Cl−挤出减少和超极化GABAA和/或甘氨酸受体介导的电流受损与癫痫，神经性疼痛和痉挛有关6，7。神经元 KCC2 通过无赖氨酸 （WNK）-STE20/SPS1 相关脯氨酸/富丙氨酸 （SPAK）/氧化应激响应 （OSR） 激酶信号传导复合物1 在其 C 末端细胞内结构域内的关键调节残基磷酸化而负调节，这有助于维持未成熟神经元中去极化的 GABA 活性2，8，9.WNK-SPAK/OSR1磷酸化苏氨酸残基906和1007（Thr906/Thr1007），随后下调KCC2的mRNA基因表达，导致其生理功能随之恶化8，10。然而，更重要的是，已知WNK-SPAK/OSR1激酶复合物磷酸化并抑制KCC2表达1，2，4，11，12已经是一个事实，并且抑制磷酸化Thr906 / Thr1007的激酶复合物信号通路与KCC2 mRNA基因13，14，15的表达增加有关。.重要的是要注意，通过蛋白质磷酸化调节神经元KCC2和Na + -K+-2Cl-共转运蛋白1（NKCC1）表达同时并以相反的模式1，4，16起作用。
在了解KCC2调节机制方面取得了持续和相当大的进展，认可开发使研究人员能够研究其功能和活动的技术;通过直接（评估激酶调控位点磷酸化）或间接（观察和监测GABA活性） 调查 。这里介绍的方案重点介绍了蛋白质印迹技术的应用，通过研究共转运蛋白在激酶调控位点Thr906/1007的磷酸化来研究神经元K+-Cl- 共转运蛋白KCC2的功能和活性。
蛋白质印迹是一种用于从组织或细胞样本中检测特定目标蛋白的方法。该方法首先通过电泳按大小分离蛋白质。然后将蛋白质电泳转移到固体载体（通常是膜）上，然后使用特异性抗体标记目标蛋白质。将抗体与使用比色法、化学发光法或荧光法检测的不同标签或荧光团偶联抗体偶联。这允许从蛋白质混合物中检测特定的目标蛋白质。该技术已用于表征KCCs1 的磷酸化特异性位点，并已用于鉴定抑制KCC3 Thr991 / Thr1048磷酸化的激酶抑制剂17。通过遵循该协议，可以特异性地检测细胞/组织裂解物中的总和磷酸化KCC2。原则上，通过该技术检测蛋白质偶联抗体具有很强的帮助性，因为它有助于提高对KCC2磷酸化位点合作活性的理解，从而阐明了参与其生理调节的分子机制。总蛋白表达的定量分析代表了KCC2的功能和活性。还有其他经典方法用于直接测量KCC2活性，例如铷离子和铊离子摄取测定。进一步的技术如膜片钳电生理学用于测量GABA活性;因此，间接反映活化和/或失活的KCC2，如细胞内氯离子稳态的评估所告知的。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40% acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A2917</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>Ammonium Per Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>248614</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>anti pSPAK</td>
			<td>Dundee University</td>
			<td>S670B</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-KCC2</td>
			<td>Dundee University</td>
			<td>S700C</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-KCC2 pSer940</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA5-95678</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-KCC2 pThr1007</td>
			<td>Dundee University</td>
			<td>S961C</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-KCC2 pThr906</td>
			<td>Dundee University</td>
			<td>S959C</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-mouse</td>
			<td>Cell Signalling technology</td>
			<td>66002</td>
			<td>Used as secondary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-NKCC1</td>
			<td>Dundee University</td>
			<td>S841B</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-NKCC1 pThr203/207/212</td>
			<td>Dundee University</td>
			<td>S763B</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-rabbit</td>
			<td>Cell Signalling technology</td>
			<td>C29F4</td>
			<td>Used as secondary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-sheep</td>
			<td>abcam</td>
			<td>ab6900</td>
			<td>Used as secondary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-SPAK</td>
			<td>Dundee University</td>
			<td>S669D</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-&#946;-Tubulin III</td>
			<td>Sigma-Aldrich</td>
			<td>T8578</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>Benzamine</td>
			<td>Merck UK</td>
			<td>135828</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td>Used as component of loading buffer and lysis buffer</td>
		</tr>
		<tr>
			<td>Bradford Coomasie</td>
			<td>Thermo Scientific</td>
			<td>1856209</td>
			<td>Used for lysate protein quantification</td>
		</tr>
		<tr>
			<td>Casting apparatus</td>
			<td>Atto&#160;</td>
			<td>WSE-1165W</td>
			<td>Used to run SDS-page electrophoresis</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804</td>
			<td>Used in lysate preparation</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>VWR</td>
			<td>MicroStar 17R</td>
			<td>Used for spinning samples</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650-100ML</td>
			<td>Used for cell culture experiment</td>
		</tr>
		<tr>
			<td>Dried Skimmed Milk</td>
			<td>Marvel</td>
			<td>N/A</td>
			<td>Used to make blocking buffer</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>ECL reagent</td>
			<td>Perkin Elmer</td>
			<td>ORTT755/2655</td>
			<td>Used to develop image for western blotting</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>D/0700/53</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>e4378</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply</td>
			<td>BioRad</td>
			<td>PowerPAC HC</td>
			<td>To supply power to run SDS-page electrophoresis</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>ThermoFisher</td>
			<td>E/0650DF/17</td>
			<td>Used for preparing sterilized equipments and environment</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum -&#160; heat inactivated</td>
			<td>Merck Life Sciences UK</td>
			<td>F9665</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Fumehood</td>
			<td>Walker</td>
			<td>A7277</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Gel Blotting - Whatman</td>
			<td>GE Healthcare&#160;</td>
			<td>10426981</td>
			<td>Used in western blotting to make transfer sandwich</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>15527</td>
			<td>Used to make buffers</td>
		</tr>
		<tr>
			<td>GraphPad Prism Software</td>
			<td>GraphPad Software, Inc., USA</td>
			<td>Version 6.0</td>
			<td>Used for plotting graphs and analysing data for&#160; western blotting</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Acros Organics</td>
			<td>10647282</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td>Grant</td>
			<td>QBT1</td>
			<td>Used to heat WB loading samples</td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>Merck UK</td>
			<td>12022001-1VL</td>
			<td>Cell line for culture experiment</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>Wayne Rasband and Contributors; NIH, USA&#160;</td>
			<td>ImageJ 1.53e</td>
			<td>Used to measure band intensities from western blotting images</td>
		</tr>
		<tr>
			<td>Imaging system</td>
			<td>BioRad</td>
			<td>ChemiDoc MP</td>
			<td>Used to take western blotting images</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>LEEC</td>
			<td>LEEC precision 190D</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Honeywell</td>
			<td>24137</td>
			<td>Used in casting gel for electrophoresis</td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Lithium dodecyl sulfate (LDS)</td>
			<td>Novex</td>
			<td>NP0008</td>
			<td>Used as loading buffer for western blotting</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acid&#160;</td>
			<td>Merck Life Sciences UK</td>
			<td>M7145</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5418</td>
			<td>Used for preparing lysates for WB</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioRad</td>
			<td>iMark</td>
			<td>Used for lysate protein concentration readout</td>
		</tr>
		<tr>
			<td>Microsoft Powerpoint</td>
			<td>Microsoft, USA</td>
			<td>PowerPoint2016</td>
			<td>Used to edit western blotting images</td>
		</tr>
		<tr>
			<td>Molecular Weight Marker</td>
			<td>BioRad</td>
			<td>1610373</td>
			<td>Used for western blotting</td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Thermo Fisher Scientific</td>
			<td>23030</td>
			<td>Used for cell culture experiment</td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Fisher Scientific</td>
			<td>45004091</td>
			<td>Used for western blotting</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo</td>
			<td>Seven compact s210</td>
			<td>Used to monitor pH of buffer solutions</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonylfluoride (PMSF)</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>PKC&#948; pThr505</td>
			<td>Cell Signalling technology</td>
			<td>9374</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>Sepharose Protein G</td>
			<td>Generon</td>
			<td>PG50-00-0002</td>
			<td>Used for immunoprecipitation</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Used as component of wash buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Used to prepare TBS-T buffer</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L5750</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>sodium orthovanadate</td>
			<td>Sigma-Aldrich</td>
			<td>S6508</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>sodium-&#946;-glycerophosphate</td>
			<td>Merck UK</td>
			<td>G9422</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Staurosporine (from Streptomyces sp.)</td>
			<td>Scientific Laboratory Supplies, UK</td>
			<td>S4400-1MG</td>
			<td>Used for cell culture experiment</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Scientifc Laboratory Supplies</td>
			<td>S0389</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T7024</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>Transfer Chamber</td>
			<td>BioRad</td>
			<td>1658005EDU</td>
			<td>Used in western blotting to transfer protein on membrane</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>T6066</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA Solution</td>
			<td>Merck Life Sciences UK</td>
			<td>T4049</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P3179</td>
			<td>Used as make TBS-T buffer</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Charles Austen</td>
			<td>Dymax 5</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific Industries</td>
			<td>K-550-GE</td>
			<td>Used in sample preparation</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries Ltd</td>
			<td>Vortex-Genie&#160; K-550-GE</td>
			<td>Used of mixing resolved sample</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant Instruments Ltd. (JB Academy)</td>
			<td>JBA5</td>
			<td>Used to incubate solutions</td>
		</tr>
	</tbody>
</table>

study of the functions and activities of neuronal k-cl co-transporter kcc2 using western blotting

氯化钾共转运蛋白2（KCC2）是阳离子氯化共转运蛋白（CCCs）溶质载体家族12（SLC12）的成员，仅在神经元中发现，对于Cl- 稳态的正常功能以及因此具有功能性GABA能抑制至关重要。KCC2的适当调节失败是有害的，并且与包括癫痫在内的几种神经系统疾病的流行有关。在了解KCC2调节所涉及的机制方面取得了相当大的进展，KCC2被认可为开发使研究人员能够研究其功能和活动的技术;通过直接（评估激酶调控位点磷酸化）或间接（观察和监测GABA活性） 调查 。在这里，该协议重点介绍了如何使用蛋白质印迹技术研究激酶调节位点 - Thr906 和 Thr1007 - 的 KCC2 磷酸化。还有其他经典方法可用于直接测量KCC2活性，例如铷离子和铊离子摄取测定。进一步的技术如膜片钳电生理学用于测量GABA活性;因此，间接反映活化和/或失活的KCC2，如细胞内氯离子稳态的评估所告知的。本文将简要讨论其中一些附加技术。

在这里， 图1 中给出的代表性结果研究了星形孢菌素和NEM对使用蛋白质印迹技术稳定表达KCC2b（HEKrnKCC2b）18 的HEK293细胞系中WNK-SPAK/OSR1介导的KCC2和NKCC1磷酸化的影响。有关代表性结果的全面细节在Zhang等人15中进行了讨论。与NEM类似，星形孢菌素是一种广泛的激酶抑制剂，可以增强KCC2转运活性并抑制NKCC1活性15</su...

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

本协议重点介绍了蛋白质印迹技术在研究神经元K-Cl共转运蛋白KCC2的功能和活性方面的应用。该方案描述了通过蛋白质印迹 在 激酶调节位点Thr906/1007处KCC2磷酸化的研究。此外，本文还简要强调了确认KCC2活性的其他方法。

使用蛋白质印迹法研究神经元K-Cl共转运蛋白KCC2的功能和活性

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

该协议可以通过直接评估其激酶分子大小磷酸化来帮助了解KCC2调节所涉及的机制，从而阐明其生理功能和活性。它可用于从相关分子的复杂混合物中鉴定和表征感兴趣的蛋白质等分子，即使蛋白质表达非常微小。该技术是复杂系统蛋白质分析以及识别异常组织功能或疾病的潜在机制的重要工具。演示这些程序的将是我实验室的博士生所罗门·乔西亚（Sunday Solomon Josiah）。首先在37摄氏度的珠浴中预布防所有试剂，并在珠浴中完全解冻稳定横断面的大鼠KCC2b人胚胎肾293细胞。然后将细胞从冷冻小瓶管转移到含有5毫升新鲜培养基的离心管中，并以1， 200 G离心细胞三至五分钟。使用固定在真空泵上的吸液器移液器吸出上清液后，将细胞重悬于10毫升新鲜培养基中。将悬浮液转移到10厘米的培养皿板上，使其在温度为37摄氏度和5%二氧化碳的培养箱中生长48小时。一旦细胞达到90%汇合度，通过吸出旧培养基并用两毫升PBS冲洗来分裂它们。加入两毫升胰蛋白酶，在室温下孵育约一到两分钟。使用两毫升完整培养基洗涤胰蛋白酶消化的细胞。在新菜肴中加入 9 毫升新鲜培养基。然后将旧盘中的一毫升溶液加入新盘中。将分裂细胞培养皿转移到培养箱中48小时，以达到90%以上的汇合度。在裂解缓冲液中收获之前，用二甲基亚砜、8 微摩尔星形孢菌素或 0.5 毫摩尔 N-乙基马来酰亚胺处理细胞 15 分钟。从横断面培养皿中吸出培养基，将培养皿放在冰上，并用冰冷的PBS清洗细胞。吸出PBS，并在培养皿中加入1.0毫升冰冷的裂解缓冲液。使用冷塑料细胞刮刀从培养皿底部刮下细胞后，将细胞悬液转移到放在冰上的微量离心管中，然后在4摄氏度下持续搅拌30分钟。在冷离心机中离心细胞裂解物后，将其放在冰上。将上清液收集到预冷的新鲜管中，并丢弃沉淀。将300微升蛋白G琼脂糖移液到微量离心管中。然后将溶液以500 G离心两分钟。弃去上清液后，加入500微升PBS并涡旋良好。离心后弃去上清液并重复该步骤。接下来，将一毫克抗 KCC2 苏氨酸 906 和抗 KCC2 苏氨酸 1007 抗体与 200 微升蛋白 G 琼脂糖珠混合，然后用 PBS 将体积补足至 500 微升。在振动平台或旋转轮上以四摄氏度摇晃两小时后，用PBS重复两次洗涤。对细胞裂解物进行蛋白质定量，并将一毫克细胞裂解物添加到洗涤的珠子中，并在旋转之前在端到端旋转器中孵育。用含有150毫摩尔氯化钠的PBS洗涤三次，然后用200微升PBS洗涤三次，然后将最终沉淀重悬于100微升LDS样品缓冲液中。在室温下在转子振动器中摇动试管五分钟。将它们在加热块中孵育并离心，然后使用上清液进行凝胶上样。组装蛋白质印迹铸造设备并倒入新鲜制备的8%分离凝胶以浇铸凝胶，从铸造玻璃顶部留出约两厘米的空间。向设置中加入 200 微升绝对异丙醇，使其在室温下静置 60 分钟。使用移液管除去异丙醇，并用约200微升蒸馏水仔细冲洗凝胶。将新鲜制备的6%堆积凝胶加入铸件设置后，轻轻放入孔梳中，并在室温下静置30分钟。然后将灌制凝胶固定到电泳槽中。将电泳缓冲液倒入电泳槽后，将5微升分子量标记物加载到第一个孔中，并将等量的蛋白质加载到SDS-PAGE凝胶的每个孔中。用LDS填充空孔，并在120伏下运行凝胶约90至120分钟。用含有20%甲醇的转印缓冲液重新水化硝酸纤维素膜。用转印缓冲液冲洗凝胶和膜，并将它们轻轻地铺在制备堆栈上。按以下顺序排列要转移的三明治：负极，三明治泡沫，滤纸冲洗SDS-PAGE凝胶，漂洗的硝酸纤维素膜，滤纸，夹心泡沫，正极。完成后，将组装好的三明治堆叠在传输罐中，并以 90 伏运行 90 分钟或以 30 伏运行 360 分钟。使用封闭缓冲液在室温下封闭干燥膜一小时。将膜与适当稀释的一抗和β-肌动蛋白在封闭缓冲液中孵育一小时，或在4摄氏度下孵育过夜。然后洗三次TBST，每次五分钟。将洗涤后的膜与在封闭缓冲液中稀释的二抗孵育60分钟，并重复TBST的三次洗涤。完成后，将洗涤后的膜放在成像板上。为了显影信号，在将成像板转移到成像系统进行成像之前，将通过在膜上混合等体积的每种增强化学发光试剂制备的溶液展开。用星形孢素和N-乙基马来酰亚胺或NEM处理HEK293细胞导致SPAK靶位点磷酸化减少，苏氨酸233位于T环激酶结构域，S环磷酸化位点丝氨酸373内源性表达的SPAK。Staurosporine降低了稳定表达KCC2b的HEK294细胞中丝氨酸940的磷酸化，但NEM显着增加了其磷酸化。此外，星形孢菌素降低苏氨酸505位点的磷酸化，而NEM在同一位点引起磷酸化的轻微但微不足道的增加。两种化合物对苏氨酸505位点的蛋白激酶C和丝氨酸940位点的KCC2b磷酸化的差异影响密切相关。代表性结果还表明，NEM引起总KCC2量的表达显著增加，而两种化合物处理时总NKCC1和SPAK的表达没有显著变化。此外，这两种化合物在苏氨酸233和丝氨酸373位点引起SPAK磷酸化降低，这与苏氨酸906/1007和苏氨酸203/207/212的节磷酸化以及内源性表达NKCC1相关。此外，星形孢素和NEM分别降低和增加丝氨酸940位点蛋白激酶C的磷酸化和稳定表达KCC2b，分别与星形孢素和NEM处理后苏氨酸505位点蛋白激酶C δ磷酸化的减少和增加相关。如果使用了不正确的一抗或二抗，则在成像过程中看不到条带。此外，如果使用浓度过低的抗体，信号可能不可见。该技术是蛋白质定量分析的相关工具，并允许将其用于许多科学和资源目的，包括作为有效的早期诊断工具。

Research

JoVE Journal

Biochemistry

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

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

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

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

科研

生物化学

SorLA and CLC:CLF-1-dependent Downregulation of CNTFR&#945; as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

SorLA and CLC:CLF-1-dependent Downregulation of CNTFRÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â± as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

SorLA和CLC：这表现在免疫印迹，溶酶体酶的抑制，和免疫组化CNTFRα的CLF-1依赖性下调

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

一种用于小数目造血干细胞的西方印迹协议

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

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

A11-positive ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

用斑点印迹分析 A11-positive β淀粉样寡聚物的制备与评价

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

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

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

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 ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

使用免疫增强技术的超高速西布洛特

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 ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...