这项工作得到了科学和技术部 (多数, 105-2320-010-007-MY3 至 pcc) 的资助, 从国家卫生研究所 (NHRI-EX105-10215BC 到 pcc), 从科学技术部 (最多 105-2314-b-400-019HJK) 和国家卫生研究所 (人权机构 MG-105-SP-10、人权署 MG-106-SP-10 到 HJK)。这项工作也得到了部分与国立阳明大学的手稿出版物, 以 PCC。资助者在研究设计、数据收集和分析、决定出版或对原稿的补救没有作用。

1. 杆状病毒表达结构的制备
<ol><li>
E3 连接 k-bZIP 的纯化, 将 k-bZIP19的 cDNA 克隆成双表达杆状病毒载体 (见材料表), 并带有 N 端的抗原标记。我们已经成功地使用了八标记 (在整个协议中被表示为向量标记 bZIP)。 注: bZIP 聚合酶链反应 (PCR) 克隆的 DNA 模板是在强力霉素治疗后从龙 F3H3-K-Rta BCBL-1 细胞中分离出的 RNA 的 cDNA 反向转录,20。<ol>
<li>将全长 k-bZIP cDNA19转化为具有毛棕榈油I 克隆站点的双表达载体。毛棕榈油我消化 PCR 产品和1µg 的双表达载体在37° c 为 3 h21。</li>
		<li>在0.8% 琼脂糖凝胶22上分离并提取经电泳后的消化 DNA。结扎的插入 (K-bZIP) 和载体的 T4 DNA 连接在16° c 30 分钟根据制造商的说明 (请参见材料表)。</li>
		<li>在1.5 毫升管中加入5µL 的结扎 DNA 到50µL 合格的大肠杆菌细胞 "a" (参见材料表)。冰敷30分钟。然后, 在四十五年代的42° c 水浴中孵育1.5 毫升管, 并立即将管子放在冰上3分钟。</li>
		<li>添加600µL soc 培养基 (0.5% (w/v) 酵母提取物, 2% (w/v) 胰, 10 毫米氯化钠, 2.5 毫米氯化钾, 20 毫米 MgSO4, 和 4% (w/v) 葡萄糖) 进入管和孵化在37° c 1 h。然后, 离心管在室温下为3分钟, 在 600 x g。</li>
		<li>用60µL Luria-Bertani 培养基 (lb; 1% (w/v) 胰, 0.5% (w/v) 酵母萃取物和85毫米氯化钠) 去除上清, 重颗粒, 并在含有100µg/毫升氨苄西林的 LB 琼脂板上传播。孵育琼脂板在37° c 为 16 h23,24。</li>
		<li>选择1殖民地接种5毫升的 LB 培养基中含有100µg/毫升氨苄西林和培养在37° c 为 16 h, 250 rpm 震动。然后, 根据制造商的说明, 从质粒提取试剂盒中提取质粒, bZIP 的载体 (参见材料表)。</li>
	</ol>
</li>
	<li>
通过将向量标记-k-bZIP 转化为有能力的大肠杆菌细胞 "B" (见材料表), 生成 bacmid 窝藏标记 k bZIP 的重组人。
	<ol>
<li>混合0.2 µg 载体-标记-bZIP 质粒和100µL 合格的E.大肠杆菌 "B" 在1.5 毫升的试管中, 在冰上放30分钟, 在42° c 的水浴中孵育, 在四十五年代, 立即把试管放在冰上3分钟。</li>
		<li>将900µL soc 培养基加入试管, 在37° c 下孵育4小时, 旋转 50 rpm。接下来, 采取10µL 的媒介, 添加额外的50µL s.o C 培养基在 LB 琼脂板上传播, 含有50µg/毫升卡那霉素, 7 µg/毫升庆大霉素, 10 µg/毫升四环素, 100 µg/毫升乳糖基板, 40 µg/毫升异丙β-d-1-thiogalactopyranoside (IPTG), 并在37° c 下孵育48小时的板材。</li>
		<li>在5毫升的 LB 培养基中接种一个白色菌落, 内含50µg/毫升卡那霉素, 7 µg/毫升庆大霉素, 10 µg/毫升四环素。在37° c 下孵育, 250 rpm 震动16小时。</li>
		<li>分离的重组 bacmid DNA 窝藏标记-K-bZIP, 量化, 并重新挂起浓度为1µg/µL25。</li>
	</ol>
</li>
	<li>
生成重组杆状表达标记-k-bZIP 由染1µg 重组 bacmid DNA 含有标记-钾-bZIP 到 Sf9 细胞在一个井 6-井板。
	<ol>
<li>种子 2 x 106 Sf9 单元格 (参见材料表) 在2毫升宽限期的昆虫培养基中提供10% 个胎儿牛血清 (FBS) 和1% 庆大霉素在每井6井板, 然后孵化在27° c 为 1 h。</li>
		<li>维护在格雷斯的昆虫培养基中的 Sf9 细胞的记录阶段培养, 提供 10% FBS, 1% 庆大霉素, 1% 洗涤剂 C (参见材料表) 在27° c 在轨道悬浮在140转每分钟。使用例和台盼蓝染色计数细胞;细胞存活能力应超过95%。使用 subcultivation 比率1:3 每2-3 天 (最小密度 ~ 0.5 x 106单元格/mL) 维护单元格。</li>
		<li>添加2µL bacmid DNA (1 µg/µL) 和98µL 减少血清培养基 (参见材料表) 到2毫升管。然后, 加入4µL 转染试剂 (见材料表), 并混合好。室温孵育15分钟。</li>
		<li>在每个井中加入这种转染混合物 (104 µL), 并在27° c 下孵育12-16 小时。</li>
		<li>用柔和的吸气去除疲惫的培养基, 用 10% FBS 和1% 的庆大霉素代替2毫升的新鲜格雷斯的昆虫培养基。Sf9 细胞松散地附着在平板表面, 不会被温和的渴望所干扰。</li>
	</ol>
</li>
	<li>
在转染和扩增杆状病毒后收集重组杆状病毒, 获得高效价股 (P1) 进行进一步的实验。
	<ol>
<li>72 h 在改变培养基后, 用无菌细胞提升器刮 Sf9 细胞, 在1.5 毫升管和漩涡中从每个个体中收集上清液, 十年代。</li>
		<li>离心机在4° c 为3分钟在 150 x g. 在1.5 毫升管中收集上清液作为 P0 杆状病毒和分, 每管中有1毫升杆状病毒。贮存在-80 ° c。</li>
		<li>种子 1 x 107 Sf9 细胞在 10 mL 的昆虫培养基中提供 10% FBS 和1% 庆大霉素在 10 cm 培养皿和孵育在27° c 为 1 h。</li>
		<li>为扩增杆状表达标记-K-bZIP, 增加0.5 毫升 P0 杆状病毒的种子 Sf9 细胞和孵化在27° c 48 小时。</li>
		<li>收集上清作为 P1 杆状病毒在15毫升管和存储在4° c, 长达1月。</li>
	</ol>
</li>
</ol>2. K-bZIP 的纯化
<ol><li>维护在格雷斯的昆虫培养基中的 Sf9 细胞的记录阶段培养, 提供 10% FBS, 1% 庆大霉素, 1% 洗涤剂 C 在27° c 在轨道悬浮在 140 rpm, 如步骤1.3.2 所述。</li>
	<li>在转导的当天, 将1毫升的杆状病毒含上清液 (P1) 加入到250毫升的 Sf9 细胞中, 密度为 2 x 106细胞/毫升。</li>
	<li>在27° c 的轨道振动器上孵育72小时的 Sf9 细胞, 140 rpm 震动, 然后用离心法在 2740 x g 处收集15分钟的4° c。</li>
	<li>在10毫升裂解缓冲液中去除上清和溶解细胞小球 (20 毫米 HEPES pH 7.9, 0.5 M 氯化钠, 1% 洗涤剂 A (参见材料目录), 2% 甘油, 蛋白酶抑制剂鸡尾酒) 和旋转在 50 rpm 使用悬浮搅拌机在4° c 为30分钟。</li>
	<li>离心机的裂解液在 1.5万 x g 为15分钟, 以收集上清液。</li>
	<li>纯化标记-K-bZIP, 孵化细胞裂解 (10 毫升) 与50µL 抗体标签磁性珠子在14毫升聚丙烯管, 并旋转 50 rpm 为 3 h 在4° c 使用悬浮搅拌机。以下蛋白质捕获, 颗粒的珠子在 800 x g 离心三十年代在4° c。除去大部分上清液, 再将残余的珠子悬浮在大约1毫升的体积内, 然后转移到1.5 毫升的管子进行洗涤。</li>
	<li>通过将含有珠子的管子放在磁性支架上, 清洗捕获的蛋白质, 一旦磁性珠子完全粘在管子的侧面, 就把上清液除去。</li>
	<li>
用裂解缓冲液洗涤珠四次。
	<ol>
<li>在1.5 毫升的管子中加入1毫升裂解缓冲液, 并将管子倒置10次。将1.5 毫升管放在磁性支架上, 并将上清液除去, 如2.7 所述。</li>
		<li>重复前一步骤3次。</li>
	</ol>
</li>
	<li>
一次用磷酸盐缓冲盐水 (PBS) 洗涤珠子。
	<ol>
<li>添加1毫升 PBS (137 毫米氯化钠, 2.7 毫米氯化钾, 8 毫米 Na2HPO4, 1.5 毫米 KH2PO4) 成1.5 毫升管, 并倒置管10次。</li>
		<li>把1.5 毫升管在一个磁性的立场, 然后删除上清。</li>
	</ol>
</li>
	<li>洗标记-K-bZIP 蛋白从抗体标签磁性珠子添加100µL 的150µg/毫升八稀释在 PBS 成1.5 毫升管。在室温下, 用悬浮混合器旋转10分钟, 50 rpm 的管子。然后, 将管子放回磁性支架上, 并在清洁的1.5 毫升管中收集含 K bZIP 的上清液。</li>
	<li>用 SDS-页分析1-5 µL 纯化的标记-K-bZIP 蛋白, 然后马斯亮蓝色染色26。在同一凝胶上装载2µg、1µg 和0.5 µg 牛血清白蛋白 (BSA) 样品, 并用图像处理程序 (如 ImageJ) 进行量化。通过与 BSA 标准的比较, 估计 bZIP 浓度。</li>
	<li>纯化的 K-bZIP 现在已经准备好用于每 sumo 测定。将 PBS 中的 K bZIP 稀释至最终浓度为 100 ng/µL, 并在-80 ° c 的小等分中贮存。</li>
</ol>3. sumo 测定
<ol><li>添加3µL 100 ng/µL 纯化标记-K-bZIP 到主混合的每一个体外sumo 反应。主混合成分含有1µL 蛋白缓冲, 1x sumo 缓冲液, 0.5 µL p53 蛋白 (0.5 毫克/毫升), 25 nm E1 活化酶 (具体 1), 50 nm µM E2 酶 (库存共轭, 具体), 10 nm 每种相扑肽 (µM, SUMO-2, 或 SUMO-3;股票具体: 5 µM) 到最后容量的17µL。</li>
	<li>将内容轻轻地混合, 在30° c 下孵育3小时的反应. 停止反应, 添加20µL 2x SDS-页加载缓冲区 (100 毫米三 HCL pH 6.8, 4% 十二烷基硫酸钠, 0.2% (w/v) 溴蓝色, 20% (v/v) 甘油, 200 毫米β-基) 和变性样品在95° c 为5分钟。</li>
	<li>
在 SDS 页上分离样品, 利用半干电泳转移装置将分离的蛋白质转移到 PVDF 膜上, 用 anti-p53 抗体进行印迹分析。
	<ol>
<li>在步骤2.11 中设置凝胶装置, 并将20µL 样品装入凝胶井。</li>
		<li>运行10% 凝胶在一个恒定的电流在80伏约120分钟 (直到染料带到达底部的凝胶)。电泳完成后, 关闭电源。</li>
		<li>断开凝胶装置和凝胶盒, 取出凝胶, 并将凝胶浮入半干传递缓冲液 (48 毫米三盐酸, 39 毫米甘氨酸, 20% 甲醇) 5 分钟。</li>
		<li>用另一种容器将 PVDF 膜浸泡在甲醇中1分钟。然后, 将 PVDF 从甲醇中取出, 放入半干式输送缓冲器。轻轻搅动膜5分钟。</li>
		<li>拆卸半干式电泳仪的安全盖。</li>
		<li>预湿滤纸, 并在下面的铂阳极上准备一个凝胶夹层: 滤纸、PVDF 膜、凝胶和滤纸。</li>
		<li>安全阴极板和安全盖, 然后运行 15 V 恒定电流为90分钟的印迹, 关闭电源, 断开半干设备, 并取出 PVDF 膜。</li>
		<li>阻挡缓冲的 PVDF 膜 (5% 非脂肪牛奶在 TBST 缓冲 (137 毫米氯化钠2, 20 毫米三盐酸, 0.1% 洗涤剂 B (参见材料目录), pH 7.6)) 在室温为 1 h, 以 30 rpm 摇动的轨道振动筛。</li>
		<li>杂交 anti-p53 抗体稀释的 PVDF 膜 (1:1, 000) 在 12-16 h 在4° c 的阻拦缓冲用悬浮混合器的 30 rpm 震动。</li>
		<li>将 PVDF 膜放入容器中, 放入 TBST。用 TBST 冲洗 PVDF 膜两次。</li>
		<li>将 PVDF 膜浸泡在 TBST 中30分钟, 用 45 rpm 摇动轨道振动筛。</li>
		<li>杂交用抗兔抗体结合辣根过氧化物酶 (HRP) 稀释 (1:4, 000) 在室温下为1小时的封闭缓冲膜, 用悬浮混合器进行 30 rpm 晃动。</li>
		<li>用 TBST 3 倍的3.4.10 清洗 PVDF 膜。</li>
		<li>用悬浮混合器将 PVDF 膜浸泡在 TBST 中30分钟, 45 rpm 震动。去除 TBST, 添加 PBS, 以保持在4° c 的 PVDF 膜高达12小时。</li>
		<li>混合增强的化学发光基底 (ECL 衬底) 试剂1和 2 (1:1) (请参阅材料表)。将 PVDF 膜从 PBS 中取出, 用纸巾或轻型雨刷器将其吸收多余水分, 并立即将400µL 的 ecl 试剂添加到每个膜的表面3-5 分钟. 通过短暂的印迹去除过量的 ecl 试剂, 但不允许 membrane 完全干燥。</li>
		<li>使用发光成像系统或显影胶片27来暴露印迹。</li>
	</ol>
</li>
</ol>

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作者没有透露任何潜在的利益冲突。

这里描述的体外sumo 协议通常用于确定 Ubc9 基板的 sumo 状态。使用标准协议研究相扑 E3 连接 sumo 功能的主要限制是相扑 E1 活化和 E2 共轭酶 Ubc9 的丰度, 在体外系统中最大限度地发挥相扑共轭作用。考虑到这一挑战, 我们认为, 将相扑 E1 和 E2 酶的量滴定到一个几乎无法检测到它们在 sumo 中活动的水平, 是确定相扑 E3 连接催化活性的唯一途径, 这是利用这种体外sumo 系统。根据这?...

相扑修饰最初被确定为一种可逆的后平移修饰 (PTM), 它调节蛋白质的稳定性1。除了直接共轭外, 相扑也可以通过相扑交互图案 (西姆斯)2的非共价键作用来附加到蛋白质上。类似于氨-磷酸化的分子, 窝藏 Src 同源 2 (SH2) 或酪氨酸绑定 (客运大楼) 域3,4, 相扑修改提供了一个额外的互动平台, 选择性招聘含 SIM 卡的效应蛋白5,6。除了调控信号转导外, 转录因子和染色质重塑分子的 sumo 调节基因表达7,8。对全基因组 sumo 模式的研究显示, 相扑修饰与阳性9、10或负10、11、12转录有关。规则, 部分原因是 sumo 的 paralogue 特异性。
有三主要的相扑亚型蛋白共轭存在于哺乳动物细胞中;这些包括 SUMO-1, 以及高度同源的 SUMO-2 和 SUMO-3 (称为 SUMO-2/3)13。相扑通常是共轭的赖氨酸 (K) 残留在ψKxD/E 共识主题的目标蛋白。在相扑 E3 酶的 SIM 负责的 paralogue 特异性14。与包含数以百计的 E3 酶的泛通路相比, 只有少数相扑 E3 酶确定了15。相扑 E3 酶是由属性定义的, 包括他们的能力 (i) 绑定 Ubc9, (ii) 通过 SIM 域名绑定相扑, 和 (iii) 加强相扑从 Ubc9 到基板转移。E3 连接不是绝对需要的相扑共轭16, 但提供基板和相扑-paralogue 特异性。因为通常只有一小部分的总基质蛋白是相扑修饰, 检测 SUMOylated 蛋白在体内始终是一个挑战。因此, 需要准确和重现性的分析, 以阐明相扑的精确功能。
体外sumo 测定法, 它评价纯化 Ubc9 对基质蛋白 sumo 的催化作用, 是研究相扑改装17的一种很好的检测方法。然而, 相扑 E3 连接活动在大多数情况下, 不能检测或只能检测与变异相扑连接高相扑 E3 连接活动和低基材的特殊性, 由于存在大量的 Ubc918的标准协议.该方法的总体成功与否主要取决于对纯化 Ubc9 的仔细滴定。这里描述的体外sumo 检测是从标准的 sumo 协议中修改的 (请参见材料表)。在卡波西肉瘤相关的疱疹病毒 (KSHV) 重新激活过程中, 对全球相扑的增加的观察促使我们识别出可能负责 sumo 的 E3 连接的病毒性相扑。在小规模筛选的互动蛋白的病毒相扑 E3 连接 K bZIP, p53 被认为是一个新的基板。在本议定书中, 我们详细地展示了在昆虫细胞的步骤涉及的表达和纯化野生型 K bZIP, 病毒相扑 E3 连接与 SUMO-2/3 特异性。纯化的 K-bZIP 的能力, 以提高 sumo p53, 一个众所周知的相扑基质, 是评估的存在减少量的 E1 和 E2 酶。通过这种改良的 sumo 试验, 研究人员可以可靠地定义相扑 E3 连接对 SUMOylate 小说或已知基底的能力, 这是研究蛋白质 sumo 的主要步骤。此外, 该方法有助于鉴别新的相扑 E3 酶低连接活性, 但高基底特异性。

<table border="0" cellpadding="0" cellspacing="0" style="width:853px;" width="854">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">pFastBac</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;">10359-016</td>
			<td style="width:219px;">dual expression baculovirus vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCR2.1-TOPO vector</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>PCR product vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">competent cell&#160; E. coli DH5&#945;</td>
			<td style="width:153px;">Yeastern Biotech</td>
			<td style="width:119px;">FYE678-80VL</td>
			<td>competent E. coli cells A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">E. coli DH10Bac</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;">10359-016</td>
			<td>competent E. coli cells B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">FugeneHD</td>
			<td style="width:153px;">Roche</td>
			<td style="width:119px;">04709705001</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Opti-MEM</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">31985062</td>
			<td>reduced serum media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">T4 Ligase</td>
			<td style="width:153px;">NEW England BioLabs</td>
			<td style="width:119px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">CpoI (RsrII)</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td style="width:119px;">ER0741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Bluo-gal</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td style="width:119px;">B1690</td>
			<td>galactosidase substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">IPTG</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">I6758-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Grace&#8217;s Insect Medium</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">11605094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Fetal Bovine Serum</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">10082147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Gentamicin</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15750060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Ampicillin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Kanamycin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">K0254-20ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">gentamicin</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">15710-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">tetracyclin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">87128-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">LB Broth</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.10285.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">HEPES</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">H4034-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">NaCl</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9888-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">KCl</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.04936.1000</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:363px;">Na2HPO4</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5136-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:363px;">KH2PO4</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">3246-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">sodium dodecyl sulfate</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.13760.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">&#946;-mercaptoethanol</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">TRIS (Base)</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">4109-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Non-fat milk</td>
			<td style="width:153px;">Fonterra</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">glycerol</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">2136-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Triton X-100</td>
			<td style="width:153px;">Amresco</td>
			<td style="width:119px;">0694-1L</td>
			<td>detergent A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Tween 20</td>
			<td style="width:153px;">Amresco</td>
			<td style="width:119px;">0777-500ML</td>
			<td>detergent B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Poloxamer 188 solution</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5556-100ML</td>
			<td>detergent C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Protease Inhibitor Cocktail Tablet</td>
			<td style="width:153px;">Roche</td>
			<td style="width:119px;">04 693 132 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">3x Flag peptide</td>
			<td style="width:153px;">Sigma</td>
			<td style="width:119px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-FLAG m2 Magnetic beads</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8823</td>
			<td>antibody-tagged magnetic beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">SUMOlink SUMO-1 Kit</td>
			<td style="width:153px;">Active Motif</td>
			<td style="width:119px;">40120</td>
			<td>standard SUMOylation protocol&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">SUMOlink SUMO-2/3 Kit</td>
			<td style="width:153px;">Active Motif</td>
			<td style="width:119px;">40220</td>
			<td>standard SUMOylation protocol&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">QIAquick Gel Extraction Kit</td>
			<td style="width:153px;">QIAGEN</td>
			<td style="width:119px;">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">QIAGEN Plasmid Mini Kit</td>
			<td style="width:153px;">QIAGEN</td>
			<td style="width:119px;">12123</td>
			<td>plasmid extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Polypropylene tubes</td>
			<td style="width:153px;">Falcon</td>
			<td style="width:119px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Petri Dish</td>
			<td style="width:153px;">Falcon</td>
			<td style="width:119px;">351029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Cell lifter</td>
			<td style="width:153px;">Corning</td>
			<td style="width:119px;">CNG3008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Loading tip</td>
			<td style="width:153px;">Sorenson BioScience</td>
			<td style="width:119px;">28480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">PVDF</td>
			<td style="width:153px;">PerkinElmer</td>
			<td style="width:119px;">NEF1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Blotting filter paper</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1703932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Mini slab gel apparatus (Bio-Rad Mini Protean II Cell)</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1658001 EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Pierce ECL Western Blotting</td>
			<td style="width:153px;">Thermo</td>
			<td style="width:119px;">32106</td>
			<td>ECL reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">suspension mixer</td>
			<td style="width:153px;">Digisystem laboratory instruments&#160; Inc.</td>
			<td style="width:119px;">SM-3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">orbital shaker</td>
			<td>Kansin instruments Co.</td>
			<td>OS701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">ImageQuant LAS 4000 
			biomolecular imager</td>
			<td style="width:153px;">GE Healthcare</td>
			<td style="width:119px;">28955810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Sf9</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td>B82501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-p53 antibody</td>
			<td>Cell Signaling</td>
			<td>#9282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-rabbit antibody</td>
			<td>GE Healthcare</td>
			<td>NA934-1ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro sumoylation assay to study sumo e3 ligase activity

小泛素样修饰 (相扑) 修饰是一种重要的后平移修饰 (PTM), 它主要通过调节蛋白质-蛋白质相互作用来传导信号转导。与泛素修饰类似, sumo 是由一个有序的酶级联, 包括 E1-activating 酶 (SAE1/SAE2), E2-conjugation 酶 (Ubc9) 和 E3-ligase (即, pia 家族, RanBP2 和 Pc2)。然而, 不同于泛, E3 连接是不必要的反应, 但确实提供了精度和效力的相扑共轭。sumo 修饰的蛋白质可以通过在体内通过沉淀与基底特异抗体和免疫与相扑特异抗体来鉴定。然而, 蛋白质相扑 E3 连接活动的演示需要体外重组的 sumo 分析使用纯化酶, 基质, 和相扑蛋白。由于在体外反应, 通常 SAE1/SAE2 和 Ubc9, 单独是足够的相扑共轭, 提高 sumo 的推定 E3 连接并不总是容易被发现。在这里, 我们描述一个修改过的在体外sumo 协议, 一致地识别相扑的修改使用一个体外重组系统。本文还介绍了一种病毒 SUMO-2/3 E3 连接的催化活性钾 bZIP 的分步协议。纯化的 K-bZIP 的 sumo 活动显示在 p53, 一个众所周知的相扑目标。本议定书不仅可以用来阐明新的相扑 E3 酶, 也可用于揭示其相扑 paralog 的特异性。

根据制造商提供的信息, sumo 测定中的 E1 和 E2 酶标准量分别为 50 nm 和 500 nm。最少量的 E2 共轭酶 Ubc9 能 SUMOylate p53 首先由一个体外sumo 测定。低至1/5 的数量的 Ubc9 使用的标准体外sumo 化验协议能够有效地 SUMOylate p53 (图 1A)。因此, 一半的 E1 酶的数量与1/10 的数量的 Ubc9 使用的标准协议是用于另一种体外sumo 化验。与标准...

Watch this Scientific Journal Video about In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity at JoVE.com

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

不像泛素酶, 很少有 E3 相扑酶被发现。这一修改的体外sumo 协议能够识别新的相扑 E3 酶由一个体外重建化验。

体外sumo 法研究相扑 E3 连接活动

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

in-vitro-sumoylation-assay-to-study-sumo-e3-ligase-activity

Research

JoVE Journal

Biology

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

9.3K 次观看.  National Yang-Ming University. 不像泛素酶, 很少有 E3 相扑酶被发现。这一修改的体外sumo 协议能够识别新的相扑 E3 酶由一个体外重建化验。

视频: In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

体外试验来测量磷脂酰乙醇胺甲基转移酶活

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

测量<em&gt;体外</em&gt;的酶学性质ATP酶活性

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

在体内人细胞 Rb 蛋白 sumo 的检测与分析

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

葡萄全基因组鉴定与表达的综合工作流 E3 泛素连接基因家族的 Meta 分析

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...