Name: identification of novel ck2 kinase substrates using a versatile biochemical approach
Uploaded: 2019-02-21T14:30:00
Description: Watch this Scientific Journal Video about Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach at JoVE.com

这项工作得到了宾夕法尼亚州卫生部向 t. i. s. 提供的英联邦普遍研究增强赠款的部分支持。

注: 确保所需材料可用并做好适当准备 (参见材料表)。
1. 准备工作
<ol><li>机械裂解组织样本 (1-2mg 的组织在100μl 的裂解缓冲液,表 1) 或培养的细胞 (10 厘米板, 在350μl 的裂解缓冲液中融合), 目标是为实验收集总共900μl 的样本。请注意, 此卷稍有超出下面描述的实验所需的内容。</li>
	<li>在4°c 条件下, 以 17, 500 x克的离心速度将样品向下旋转3分钟...

<ol>
	<li>Litchfield, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+CK2:+structure,+regulation+and+role+in+cellular+decisions+of+life+and+death.">Protein kinase CK2: structure, regulation and role in cellular decisions of life and death.</a> Biochemical Journal. 369 (Pt 1), 1-15 (2003).</li><li>Ahmed, K., Gerber, D. A., Cochet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Joining+the+cell+survival+squad:+an+emerging+role+for+protein+kinase+CK2).">Joining the cell survival squad: an emerging role for protein kinase CK2).</a> Trends in Cell Biology. 12 (5), 226-230 (2002).</li><li>Meggio, F., Pinna, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-thousand-and-one+substrates+of+protein+kinase+CK2.">One-thousand-and-one substrates of protein kinase CK2.</a> The FASEB Journal. 17 (3), 349-368 (2003).</li><li>Niefind, K., Guerra, B., Ermakowa, I., Issinger, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+human+protein+kinase+CK2:+insights+into+basic+properties+of+the+CK2+holoenzyme.">Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme.</a> The EMBO Journal. 20 (19), 5320-5331 (2001).</li><li>Sarno, S., Ghisellini, P., Pinna, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+activation+mechanism+of+protein+kinase+CK2.+The+N-terminal+segment+is+essential+for+constitutive+activity+of+the+catalytic+subunit+but+not+of+the+holoenzyme.">Unique activation mechanism of protein kinase CK2. The N-terminal segment is essential for constitutive activity of the catalytic subunit but not of the holoenzyme.</a> Journal of Biological Chemistry. 277 (25), 22509-22514 (2002).</li><li>Niefind, K., Putter, M., Guerra, B., Issinger, O. G., Schomburg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTP+plus+water+mimic+ATP+in+the+active+site+of+protein+kinase+CK2.">GTP plus water mimic ATP in the active site of protein kinase CK2.</a> Nature Structural &amp; Molecular Biology. 6 (12), 1100-1103 (1999).</li><li>Lou, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alpha+catalytic+subunit+of+protein+kinase+CK2+is+required+for+mouse+embryonic+development.">The alpha catalytic subunit of protein kinase CK2 is required for mouse embryonic development.</a> Molecular and Cellular Biology. 28 (1), 131-139 (2008).</li><li>Buchou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+regulatory+beta+subunit+of+protein+kinase+CK2+in+mice+leads+to+a+cell-autonomous+defect+and+early+embryonic+lethality.">Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality.</a> Molecular and Cellular Biology. 23 (3), 908-915 (2003).</li><li>Chua, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CK2+in+Cancer:+Cellular+and+Biochemical+Mechanisms+and+Potential+Therapeutic+Target.">CK2 in Cancer: Cellular and Biochemical Mechanisms and Potential Therapeutic Target.</a> Pharmaceuticals (Basel). 10 (1), (2017).</li><li>Tawfic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+CK2+signal+in+neoplasia.">Protein kinase CK2 signal in neoplasia.</a> Histology and Histopathology. 16 (2), 573-582 (2001).</li><li>Hanif, I. M., Hanif, I. M., Shazib, M. A., Ahmad, K. A., Pervaiz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Casein+Kinase+II:+an+attractive+target+for+anti-cancer+drug+design.">Casein Kinase II: an attractive target for anti-cancer drug design.</a> The International Journal of Biochemistry &amp; Cell Biology. 42 (10), 1602-1605 (2010).</li><li>Siddiqui-Jain, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CX-4945,+an+orally+bioavailable+selective+inhibitor+of+protein+kinase+CK2,+inhibits+prosurvival+and+angiogenic+signaling+and+exhibits+antitumor+efficacy.">CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy.</a> Cancer Research. 70 (24), 10288-10298 (2010).</li><li>Chon, H. J., Bae, K. J., Lee, Y., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+casein+kinase+2+inhibitor,+CX-4945,+as+an+anti-cancer+drug+in+treatment+of+human+hematological+malignancies.">The casein kinase 2 inhibitor, CX-4945, as an anti-cancer drug in treatment of human hematological malignancies.</a> Frontiers in Pharmacology. 6, 70 (2015).</li><li>Perea, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIGB-300,+a+novel+proapoptotic+peptide+that+impairs+the+CK2+phosphorylation+and+exhibits+anticancer+properties+both+in+vitro+and+in+vivo.">CIGB-300, a novel proapoptotic peptide that impairs the CK2 phosphorylation and exhibits anticancer properties both in vitro and in vivo.</a> Molecular and Cellular Biochemistry. (1-2), 163-167 (2008).</li><li>Xiao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+expression+of+nucleolin+phosphorylation-deficient+mutant+confers+dominant-negative+effect+on+cell+proliferation.">Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation.</a> PLoS One. 9 (10), e109858 (2014).</li><li>Shi, Y., Brown, E. D., Walsh, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+recombinant+human+casein+kinase+II+and+recombinant+heat+shock+protein+90+in+Escherichia+coli+and+characterization+of+their+interactions.">Expression of recombinant human casein kinase II and recombinant heat shock protein 90 in Escherichia coli and characterization of their interactions.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (7), 2767-2771 (1994).</li><li>Mollapour, M., Tsutsumi, S., Kim, Y. S., Trepel, J., Neckers, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Casein+kinase+2+phosphorylation+of+Hsp90+threonine+22+modulates+chaperone+function+and+drug+sensitivity.">Casein kinase 2 phosphorylation of Hsp90 threonine 22 modulates chaperone function and drug sensitivity.</a> Oncotarget. 2 (5), 407-417 (2011).</li><li>McMillan, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+kinase+CK2+substrate+Jabba+modulates+lipid+metabolism+during+Drosophila+oogenesis.">The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis.</a> Journal of Biological Chemistry. 293 (8), 2990-3002 (2018).</li><li>Turowec, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+CK2+is+a+constitutively+active+enzyme+that+promotes+cell+survival:+strategies+to+identify+CK2+substrates+and+manipulate+its+activity+in+mammalian+cells.">Protein kinase CK2 is a constitutively active enzyme that promotes cell survival: strategies to identify CK2 substrates and manipulate its activity in mammalian cells.</a> Methods in Enzymology. , 471-493 (2010).</li><li>Rusin, S. F., Adamo, M. E., Kettenbach, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Candidate+Casein+Kinase+2+Substrates+in+Mitosis+by+Quantitative+Phosphoproteomics.">Identification of Candidate Casein Kinase 2 Substrates in Mitosis by Quantitative Phosphoproteomics.</a> Frontiers in Cell and Developmental Biologyl. 5, 97 (2017).</li><li>Bian, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+screening+of+CK2+kinase+substrates+by+an+integrated+phosphoproteomics+workflow.">Global screening of CK2 kinase substrates by an integrated phosphoproteomics workflow.</a> Scientific Reports. 3, 3460 (2013).</li><li>Franchin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+a+phosphoproteome+readily+altered+by+the+protein+kinase+CK2+inhibitor+quinalizarin+in+HEK-293T+cells.">Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.</a> Biochimica et Biophysica Acta. 1854 (6), 609-623 (2015).</li><li>Franchin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-evaluation+of+protein+kinase+CK2+pleiotropy:+new+insights+provided+by+a+phosphoproteomics+analysis+of+CK2+knockout+cells.">Re-evaluation of protein kinase CK2 pleiotropy: new insights provided by a phosphoproteomics analysis of CK2 knockout cells.</a> Cellular and Molecular Life Sciences. 75 (11), 2011-2026 (2018).</li></ol>

在这里, 我们描述了一种相对简单的生化方法来识别复杂生物样本中的蛋白激酶 ck2 底物。该协议的关键步骤是基于 ck2 的不寻常酶特性, 包括 ck2 依赖硫代化特定底物蛋白使用 gtp γ-s 及其随后的免疫沉淀和鉴定。通过这些结果, 我们已经证明了这种方法的效用和多功能性, 因为我们现在已经将这一策略应用于人类胶质母细胞瘤细胞和食子细胞瘤卵巢18。
<p class="jove_...

蛋白激酶是信号转导级联的关键成分。这些酶对底物蛋白的磷酸化产生生物反应, 调节控制细胞分裂、代谢和分化等关键事件的关键事件。ck2 是一种普遍表达的嗜酸性血清-苏氨酸激酶, 从酵母保存到人类, 在许多细胞过程中发挥着重要作用, 从转录调节到细胞周期进展到细胞凋亡1 ,2,3。酶是由两个催化α (或α ') 亚基和两个调节β亚基4组成的异体体.ck2 除了具有高度的增压外, 还表现出另外两个使其分析复杂化的不寻常特征, 即本构活性5和双共底物特异性 6。后一种特性赋予 ck2 使用 gtp 以及 atp 对底物蛋白进行磷酸化的能力。
小鼠 ck2 催化或调控亚基的遗传缺失导致胚胎死亡, 表明其在发育和器官发生过程中起着至关重要的作用7,8。ck2 在几种癌症中也有过度表达, 因此是一个很有希望的治疗目标9、10、11.事实上, 目前正在为此目的研究针对 ck2 激酶活性的特定抑制剂12、13、14.虽然抑制 ck2 是一个可行的选择, 但鉴于其多向异性, 另一种也许更合理的办法是针对某些癌症进展的关键 ck2 基板。因此, ck2 底物蛋白的综合鉴定和鉴定对于阐明这种激酶在特定组织或肿瘤类型中的特异性功能将具有重要意义。
在这里, 我们描述了一种通用的生化方法, 用于识别 ck2 底物从复杂的生物样本, 如细胞或组织裂解液。该协议利用 ck2 的双共底物特性, 利用 gtp 模拟 gtpγs (鸟苷 5 '-[γ-thio] 三磷酸), 而其他内源激酶不能使用。这有效地允许激酶在此样本中 "标记" 其底物, 以便随后进行隔离和识别。

<table border="0" cellpadding="0" cellspacing="0" style="width:2010px;" width="2008">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">12 mg/mL PNBM</td>
			<td style="width:599px;">Abcam</td>
			<td style="width:599px;">ab138910</td>
			<td style="width:406px;">40.5 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.5 mM GTP&#947;S</td>
			<td>Sigma-Aldrich</td>
			<td>G8634-1MG</td>
			<td>5.4 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CK2&#945; (E-7) mouse monoclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-373894</td>
			<td>1:1000 for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GAPDH (6C5) mouse monoclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32233</td>
			<td>1:1000 for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-nucleolin rabbit polyclonal antibody</td>
			<td>Abcam</td>
			<td style="width:599px;">ab22758</td>
			<td>1:1000 for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-thiophosphate ester [51-8] rabbit monoclonal antibody</td>
			<td>Abcam</td>
			<td style="width:599px;">ab92570</td>
			<td>Varies (final concentration 2.8 &#181;g for each sample)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge pre-set to 4&#186;C</td>
			<td>ThermoScientific</td>
			<td>Sorvall Legend Micro 21R Cat# 75-772-436&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cOmplete Mini EDTA-Free Protease Inhibitor</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysis Buffer</td>
			<td>See recipe below</td>
			<td>See recipe below</td>
			<td>30 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal rabbit IgG antibody (isotype control)</td>
			<td>Cell Signaling Technology</td>
			<td>2729S&#160;</td>
			<td>Varies (final concentration 2.8 &#181;g for each sample)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD MiniTrap Column</td>
			<td>GE Healthcare</td>
			<td>28-9180-10</td>
			<td>3 columns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein A/G Plus Agarose Beads</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2003</td>
			<td>600 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant human CK2 holoenzyme</td>
			<td>New England Biolabs</td>
			<td>P6010S</td>
			<td>2.7 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotator</td>
			<td>Labnet: Mini Labroller</td>
			<td>Mini Labroller SKU# H5500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T98G human glioblastoma cells</td>
			<td>ATCC</td>
			<td>CRL-1690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath pre-set to 30&#186;C</td>
			<td>Shel Lab</td>
			<td>H20 Bath Series Model# SWB15</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of novel ck2 kinase substrates using a versatile biochemical approach

对激酶-底物关系的研究对于全面了解这些酶及其下游目标在生理和病理状态下的功能至关重要。ck2 是一种进化保守的血清/苏氨酸激酶, 在多个细胞过程中含有越来越多的数百种底物。由于其各向异性的特性, 识别和表征一套完整的 ck2 基板特别具有挑战性, 仍然是研究这一重要酶的一个障碍。为了应对这一挑战, 我们设计了一种通用的实验策略, 能够有针对性地丰富和识别假定的 ck2 基板。该协议利用了 ck2 独特的双基底物特异性, 允许其底物在细胞或组织裂解液中进行特定的硫代磷酸化。这些底物蛋白随后被烷基化, 免疫沉淀, 并通过液相色谱-串联质谱 (lc-msn) 进行鉴定。我们以前使用这种方法成功地识别 ck2 底物从嗜酸性粒细胞卵巢, 在这里我们扩大了该协议的应用, 人类胶质母细胞瘤细胞, 说明了这种方法的适应性, 以研究这种激酶在各种模型生物和实验系统中的生物学作用。

图 1提供了实验过程的示意图。该技术的基础是 ck2 在磷基转移中使用 gtp 的异常能力。在外源 ck2 全酶与 gtp 类似物 gtpγs 一起添加到细胞裂解液中, 可导致内源性 ck2 底物的硫代磷酸化。随后用烷基化试剂对苯甲磺酸对硝基苯 (pnbm) 处理裂解液, 在这些特定底物蛋白上生成硫代磷酸酯, 然后使用抗硫代磷酸酯抗体进行免疫沉淀并最终通过质谱技术...

Watch this Scientific Journal Video about Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach at JoVE.com

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

该协议的目的是标记、丰富和识别复杂生物样品 (如细胞裂解物或组织均质) 中的蛋白激酶 ck2 底物。此方法利用 ck2 生物学的独特方面来实现这一目的。

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

该协议允许从复杂的生物样品（如细胞或组织脱盐）中对内源性CK2基质进行特定标记和随后的浓缩和鉴定。此方法可应用于任何细胞类型或组织。从而促进各种生物学背景下的CK2研究。演示这个程序的将是约翰·乔伊诺夫斯基，一个研究生，从我的实验室。首先，机械地对组织样本或培养细胞进行培养，如文本协议中所述，总共收集 900 微升样品。在17，500倍重力下离心，在4摄氏度下离心3分钟。然后，将270微升的上流液转移到三个新的1.7毫升微离心管中。取出 40 微升的剩余上一升用作输入控制，并将其传输到新管。将所有样品放在冰上。首先，标记三个样品管，如下所示。在激酶反应管上，加入2.7微升的CK2和2.7微升的2.5毫摩尔GPGAmmaS。轻拂管子混合，并立即放在冰上。在GPGAmmaS仅管，添加2.7微升2.5毫摩尔GPGAmmaS，和2.7微升的Lysis缓冲液。轻拂这个管子混合，并立即把它放在冰上。到PNBM仅管添加5.4微升的透析缓冲液。轻拂管子混合，并立即放在冰上。在30摄氏度的水浴中孵育所有三个管子一分钟。在此之后，以每毫升12毫克的速度加入13.5微升的PNBM。反转管子混合样品，在室温下孵育样品一小时。当样本孵育时，标记要加载样本的每个列，如下所示。通过多次反转每列来准备列，以重新在存储缓冲区中重新填充 Sephadex G-25 树脂。将每一柱连接到夹紧支架上，让其不受干扰地坐约五分钟，让树脂沉淀。接下来，在每个柱的底部开口下方放置一个管。从每列的顶部和底部拆下盖，使存储缓冲液通过重力排入管中。存储缓冲区耗尽后，向每列添加大约 2.7 毫升的 Lysis 缓冲区，以平衡它们，确保收集和丢弃流。重复此过程添加 Lysis 缓冲区并丢弃流通过三次。首先，将每个样本加载到各自的列中。收集并丢弃流。将 420 微升的 Lysis 缓冲液添加到每列中，以清洗样品。让 Lysis 缓冲区通过列进行筛选，并收集和丢弃流通过。然后，将管放在每个列下的位置以进行样品收集。将 500 微升的 Lysis 缓冲液添加到每列中，以对样品进行采样。收集流经，现在含有丰富的硫磷和alkylat CK2基质在激酶反应。GTPgammaS 仅反应将包含硫磷化基质，由任何内源 CK2 存在。PNMB唯一反应将包含背景alkylat蛋白。作为"洗脱输入控制"样品，从每个洗脱中去除 80 微升。接下来，将每个样品分成两个管，每个管包含 200 微升。将每个管标为抗硫磷酸盐酯或免疫球蛋白 G，如下所示。在每个抗硫磷酯管中加入2.8微克的抗硫磷酯抗体。在每个 IGG 管中加入 2.8 微克异型控制抗体。然后将管子放在四摄氏度的旋转器上两个小时。在样品孵育的最后15分钟，使用干净的剃须刀刀片切断P200移液器尖端的末端，以增加仪表尺寸。使用前，短暂旋转含有红玫瑰珠的存储管。使用切割移液器尖端，将每个免疫沉淀的珠浆的 100 微升转移到新的 1.7 毫升微离心管中。在每次转移之前旋转珠子，以防止珠子沉降，这一点很重要。在17，500倍的重力和4摄氏度的离心管下离心一分钟。取出并丢弃上一液。通过添加 200 微升的 Lysis 缓冲液和短暂涡旋混合来重新填充珠子。重复离心和洗涤珠子三次的过程。最后洗涤后，将珠子放在冰上，直到准备好继续。当样品孵育完成后，在17，500倍重力和4摄氏度的温度下离心3分钟。然后，将每个样品的200微升转移到含有洗涤珠子的管子中。将管子放在四摄氏度的旋转器上一小时。接下来，在17，500倍的重力和4摄氏度的离心管下离心1分钟。从每个上一液中去除 40 微升，以节省消耗控制。取出并丢弃上一液的剩余部分，小心不要打扰珠子。通过在每个样品中加入200微升的Lysis缓冲液来清洗样品，并短暂地旋转。在17，500倍重力和4摄氏度的离心1分钟，丢弃上一代。重复此洗涤和离心过程三次，注意不打扰珠子。在此之后，向每个含有珠子的样品添加 50 微升 2X 样品缓冲液。对于所有其他样本（包括输入控制、洗脱输入控件和损耗控制），添加 8 个 6X 样本缓冲区的微升。除含有珠子的管子外，每个管上下移液混合，并在 95 摄氏度下加热所有样品五分钟，然后继续使用 SDS-PAGE。要评估免疫沉淀是否成功，在单独的 12.5% 聚丙烯酰胺凝胶上运行从珠子中分离出的每个样品的 25 到 30 微升。用库马西蓝染色每个凝胶，以可视化实验协议不同阶段的富集蛋白质。使用新的，干净的剃须刀刀片，小心切除任何独特的波段存在于激酶反应抗硫磷酸盐酯IP巷，同时记录其近似的分子量。每个独特的频带都应该使用新的剃须刀刀片。该技术的基础是该技术是CK2使用GTP进行磷酸组转移的异常能力。将外源性CK2全息酶与GTP类似GTPPgamas添加到细胞落酸中，导致内源性CK2基质的硫磷化。随后用alkylating试剂p-Nitrobenzyl中酶酸酯随后处理这些特异性基质蛋白产生硫磷酸盐酯酸酯，然后使用抗硫磷酯酯抗体进行免疫沉淀，最终由质谱法鉴定。在此显示的代表性阳性结果中，CK2 依赖性、硫磷化和随后的烷基化是成功的。不出所料，仅在含有完整激酶反应的车道上观察到西印的增强抗硫磷酯信号，而仅在GTPgammaS中观察到，PNMB只处理样品。免疫沉淀和暗示蛋白的库马西蓝色染色凝胶，也显示出积极的结果。由于多个独特的波段仅在抗硫磷酸盐酯IP巷中可见，其中酸盐通过外源性CK2和GTPgamas孵育。标记带然后从凝胶中切除，并提交质谱仪进行蛋白质鉴定。按照此程序，必须通过附加方法（例如在标准体外激酶测定中通过 CK2 依赖磷酸化）验证质谱仪所识别的 CK2 基质基质。

Research

JoVE Journal

Biochemistry

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

科研

生物化学

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Assaying Protein Kinase Activity with Radiolabeled ATP

用放射性标记的ATP测定蛋白激酶活性

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

通过对Rho激酶活性的暂时抑制,增强人类诱导多能干细胞衍生心肌细胞的诱导

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...