产品分类
EIF2AK1 Knockout Raji Cell Pool
- 产品描述
- 细胞复苏
- 细胞传代
- 细胞冻存
- 抗体验证结果
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- 品牌: ELEM粒曼
- 商品名称: EIF2AK1 Knockout Raji Cell Pool
- 商品编号: LM01900086232
- Gene Symbol: EIF2AK1
- Ensembl ID: ENSG00000086232
- Uniprot ID: Q9BQI3
- 宿主细胞 / 类型: Raji/人Burkitt's淋巴瘤细胞
- NCBI Gene ID: 27102
- 规格: 1×10^6 cells/ 冻存管
- 筛选标记: N/A
- 生长特性: 悬浮细胞
- 培养条件: 37℃,5% CO2 的培养箱，1/2 到 1/4 传代
- 倍增时间: ~24-36 hours
- 生长培养基: RPMI-1640＋10% FBS＋1% P/S
- 参考换液频率: 2-3天换液
- 支原体检测结果: 阴性
- 敲除效率（Sanger测序）: >70%
- 蛋白质组验证结果: N/A
- 抗体货号: 添加中
- 目标基因介绍: Metabolic-stress sensing protein kinase that phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (EIF2S1/eIF-2-alpha) in response to various stress conditions (PubMed:32132706, PubMed:32132707). Key activator of the integrated stress response (ISR) required for adaptation to various stress, such as heme deficiency, oxidative stress, osmotic shock, mitochondrial dysfunction and heat shock (PubMed:32132706, PubMed:32132707). EIF2S1/eIF-2-alpha phosphorylation in response to stress converts EIF2S1/eIF-2-alpha in a global protein synthesis inhibitor, leading to a global attenuation of cap-dependent translation, while concomitantly initiating the preferential translation of ISR-specific mRNAs, such as the transcriptional activator ATF4, and hence allowing ATF4-mediated reprogramming (PubMed:32132706, PubMed:32132707). Acts as a key sensor of heme-deficiency: in normal conditions, binds hemin via a cysteine thiolate and histidine nitrogenous coordination, leading to inhibit the protein kinase activity (By similarity). This binding occurs with moderate affinity, allowing it to sense the heme concentration within the cell: heme depletion relieves inhibition and stimulates kinase activity, activating the ISR (By similarity). Thanks to this unique heme-sensing capacity, plays a crucial role to shut off protein synthesis during acute heme-deficient conditions (By similarity). In red blood cells (RBCs), controls hemoglobin synthesis ensuring a coordinated regulation of the synthesis of its heme and globin moieties (By similarity). It thereby plays an essential protective role for RBC survival in anemias of iron deficiency (By similarity). Similarly, in hepatocytes, involved in heme-mediated translational control of CYP2B and CYP3A and possibly other hepatic P450 cytochromes (By similarity). May also regulate endoplasmic reticulum (ER) stress during acute heme-deficient conditions (By similarity). Also activates the ISR in response to mitochondrial dysfunction: HRI/EIF2AK1 protein kinase activity is activated upon binding to the processed form of DELE1 (S-DELE1), thereby promoting the ATF4-mediated reprogramming (PubMed:32132706, PubMed:32132707).
- 细胞开发路径: 采用CRISPR-RNP方法生成稳定KO Cell Pool；Sanger 测序结果显示KO Cell Pool敲除效率>70%。
- 应用: 高敲除效率的基因敲除细胞池（KO Cell Pool），特别适用于初步功能分析、复杂疾病模型的开发、精准药物筛选以及广泛的基因发现研究。KO pool能够无需繁琐的单克隆挑选过程，直接应用于多种类型的测定和分析，大幅提升实验效率。
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01. 在 37℃水浴中预热完全培养基。
02. 将冻存管在 37℃水浴中解冻 1-2 分钟。
03. 将冻存管转移到生物安全柜中，并用 70% 乙醇擦拭表面。
04. 拧开冻存管管盖，将细胞悬液轻轻转移到含有 9mL 完全培养基的无菌离心管中。
05. 在室温下以 125g 离心 5-7 分钟，弃上清。
06. 用 5mL 的完整培养基重悬细胞沉淀，将细胞悬液转移到 T25 培养瓶中。
07. 将细胞转移到 37℃,5% CO2 的培养箱中培养。
08. 参考传代比例：1/2 到 1/4 传代，2-3 天长满。 -
01. 取少量细胞悬液进行细胞计数及活力检测，当细胞密度达到1.5x10^6 cells/mL时，可进行细胞传代。
02. 将培养基从 4℃冰箱中拿出， 置于 37℃水浴中温度接近 37℃时取出并在瓶子表面喷洒 75% 酒精后置于生物安全柜中。03. 从培养箱中取出待传代的培养瓶，瓶身喷洒 75% 酒精后置于生物安全柜中。
04. 取足量细胞加入盛有新鲜培养基的培养瓶中，按需求调整接种比例，并补充培养瓶中完全培养基，T75 加至 13-15mL，T25 加至 5mL。将细胞密度维持在7x10^5 cells/mL。
05. 盖上瓶盖拧紧后轻轻晃动瓶身，使细胞混合均匀后置于 37℃,5% CO2 培养箱中。
-
01. 准备冻存液，并提前预冷。
02. 确保待冻存的细胞满足冻存要求，用显微镜检查以下状态：健康的外观及形态特征、所处生 长周期（对数晚期）、无污染或衰退迹象。
03. 将细胞收集到无菌的锥形离心管中并计数细胞。
04. 在室温下以250×g离心细胞5分钟，并小心吸出培养基。
05. 将细胞以至少2x106细胞/mL的密度重悬于冻存液中。
06. 吹打均匀后按照每管 1mL 的量分装至冻存管。
07. 将细胞放在程序降温盒中，在 -80℃冰箱中冷冻。
08. 后续将细胞转移到液氮罐中，以便长期储存。 - 抗体验证中
产品类型： 基因敲除细胞池
细胞系信息
Gene Symbol
EIF2AK1
NCBI Gene ID
27102
Ensembl ID
ENSG00000086232
Uniprot ID
Q9BQI3
筛选标记
N/A
宿主细胞 / 类型
Raji/人Burkitt's淋巴瘤细胞
规格
1×10^6 cells/ 冻存管
生长培养基
RPMI-1640＋10% FBS＋1% P/S
生长特性
悬浮细胞
培养条件
37℃,5% CO2 的培养箱，1/2 到 1/4 传代
倍增时间
~24-36 hours
参考换液频率
2-3天换液
支原体检测结果
阴性
敲除验证
敲除效率（Sanger测序）
>70%
蛋白质组验证结果
N/A
抗体货号
添加中
抗体验证结果
细胞系说明
目标基因介绍
Metabolic-stress sensing protein kinase that phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (EIF2S1/eIF-2-alpha) in response to various stress conditions (PubMed:32132706, PubMed:32132707). Key activator of the integrated stress response (ISR) required for adaptation to various stress, such as heme deficiency, oxidative stress, osmotic shock, mitochondrial dysfunction and heat shock (PubMed:32132706, PubMed:32132707). EIF2S1/eIF-2-alpha phosphorylation in response to stress converts EIF2S1/eIF-2-alpha in a global protein synthesis inhibitor, leading to a global attenuation of cap-dependent translation, while concomitantly initiating the preferential translation of ISR-specific mRNAs, such as the transcriptional activator ATF4, and hence allowing ATF4-mediated reprogramming (PubMed:32132706, PubMed:32132707). Acts as a key sensor of heme-deficiency: in normal conditions, binds hemin via a cysteine thiolate and histidine nitrogenous coordination, leading to inhibit the protein kinase activity (By similarity). This binding occurs with moderate affinity, allowing it to sense the heme concentration within the cell: heme depletion relieves inhibition and stimulates kinase activity, activating the ISR (By similarity). Thanks to this unique heme-sensing capacity, plays a crucial role to shut off protein synthesis during acute heme-deficient conditions (By similarity). In red blood cells (RBCs), controls hemoglobin synthesis ensuring a coordinated regulation of the synthesis of its heme and globin moieties (By similarity). It thereby plays an essential protective role for RBC survival in anemias of iron deficiency (By similarity). Similarly, in hepatocytes, involved in heme-mediated translational control of CYP2B and CYP3A and possibly other hepatic P450 cytochromes (By similarity). May also regulate endoplasmic reticulum (ER) stress during acute heme-deficient conditions (By similarity). Also activates the ISR in response to mitochondrial dysfunction: HRI/EIF2AK1 protein kinase activity is activated upon binding to the processed form of DELE1 (S-DELE1), thereby promoting the ATF4-mediated reprogramming (PubMed:32132706, PubMed:32132707).
细胞开发路径
采用CRISPR-RNP方法生成稳定KO Cell Pool；Sanger 测序结果显示KO Cell Pool敲除效率>70%。
应用
高敲除效率的基因敲除细胞池（KO Cell Pool），特别适用于初步功能分析、复杂疾病模型的开发、精准药物筛选以及广泛的基因发现研究。KO pool能够无需繁琐的单克隆挑选过程，直接应用于多种类型的测定和分析，大幅提升实验效率。
细胞培养说明
细胞复苏
01. 在 37℃水浴中预热完全培养基。
02. 将冻存管在 37℃水浴中解冻 1-2 分钟。
03. 将冻存管转移到生物安全柜中，并用 70% 乙醇擦拭表面。
04. 拧开冻存管管盖，将细胞悬液轻轻转移到含有 9mL 完全培养基的无菌离心管中。
05. 在室温下以 125g 离心 5-7 分钟，弃上清。
06. 用 5mL 的完整培养基重悬细胞沉淀，将细胞悬液转移到 T25 培养瓶中。
07. 将细胞转移到 37℃,5% CO2 的培养箱中培养。
08. 参考传代比例：1/2 到 1/4 传代，2-3 天长满。
细胞传代
01. 取少量细胞悬液进行细胞计数及活力检测，当细胞密度达到1.5x10^6 cells/mL时，可进行细胞传代。
02. 将培养基从 4℃冰箱中拿出， 置于 37℃水浴中温度接近 37℃时取出并在瓶子表面喷洒 75% 酒精后置于生物安全柜中。
03. 从培养箱中取出待传代的培养瓶，瓶身喷洒 75% 酒精后置于生物安全柜中。
04. 取足量细胞加入盛有新鲜培养基的培养瓶中，按需求调整接种比例，并补充培养瓶中完全培养基，T75 加至 13-15mL，T25 加至 5mL。将细胞密度维持在7x10^5 cells/mL。
05. 盖上瓶盖拧紧后轻轻晃动瓶身，使细胞混合均匀后置于 37℃,5% CO2 培养箱中。
细胞冻存
01. 准备冻存液，并提前预冷。
02. 确保待冻存的细胞满足冻存要求，用显微镜检查以下状态：健康的外观及形态特征、所处生 长周期（对数晚期）、无污染或衰退迹象。
03. 将细胞收集到无菌的锥形离心管中并计数细胞。
04. 在室温下以250×g离心细胞5分钟，并小心吸出培养基。
05. 将细胞以至少2x106细胞/mL的密度重悬于冻存液中。
06. 吹打均匀后按照每管 1mL 的量分装至冻存管。
07. 将细胞放在程序降温盒中，在 -80℃冰箱中冷冻。
08. 后续将细胞转移到液氮罐中，以便长期储存。