产品分类
EP300 HiBiT HEK293 Harbor™ Cell Line
- 产品描述
- 细胞复苏
- 细胞传代
- 细胞冻存
- 验证数据
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- 商品名称: EP300 HiBiT HEK293 Harbor™ Cell Line
- 商品编号: LMH01024100393
- 物种: human
- 细胞形态: 多边形，上皮样细胞，贴壁
- 规格: 冻存管1×10⁶/管或T25活细胞/瓶，贴壁细胞汇合度70%以上，悬浮细胞量1×10⁶/瓶
- 完全培养基成分: MEM+10%FBS+1%P/S
- 抗性基因: puro，2ug/ml
- 培养环境: 37℃,5% CO2 的培养箱，1/3到 1/5传代
- 传代比例: 1/3-1/5
- 传代频次: 2-3天
- 支原体检测: 阴性
- 靶标蛋白及功能: (Microbial infection) In case of HIV-1 infection, it is recruited by the viral protein Tat. Regulates Tat's transactivating activity and may help inducing chromatin remodeling of proviral genes. Binds to and may be involved in the transforming capacity of the adenovirus E1A protein.||Functions as histone acetyltransferase and regulates transcription via chromatin remodeling (PubMed:23415232, PubMed:23934153, PubMed:8945521). Acetylates all four core histones in nucleosomes. Histone acetylation gives an epigenetic tag for transcriptional activation (PubMed:23415232, PubMed:23934153, PubMed:8945521). Mediates cAMP-gene regulation by binding specifically to phosphorylated CREB protein. Mediates acetylation of histone H3 at 'Lys-122' (H3K122ac), a modification that localizes at the surface of the histone octamer and stimulates transcription, possibly by promoting nucleosome instability. Mediates acetylation of histone H3 at 'Lys-27' (H3K27ac) (PubMed:23911289). Also functions as acetyltransferase for non-histone targets, such as ALX1, HDAC1, PRMT1 or SIRT2 (PubMed:12929931, PubMed:16762839, PubMed:18722353). Acetylates 'Lys-131' of ALX1 and acts as its coactivator (PubMed:12929931). Acetylates SIRT2 and is proposed to indirectly increase the transcriptional activity of TP53 through acetylation and subsequent attenuation of SIRT2 deacetylase function (PubMed:18722353). Acetylates HDAC1 leading to its inactivation and modulation of transcription (PubMed:16762839). Acetylates 'Lys-247' of EGR2 (By similarity). Acts as a TFAP2A-mediated transcriptional coactivator in presence of CITED2 (PubMed:12586840). Plays a role as a coactivator of NEUROD1-dependent transcription of the secretin and p21 genes and controls terminal differentiation of cells in the intestinal epithelium. Promotes cardiac myocyte enlargement. Can also mediate transcriptional repression. Acetylates FOXO1 and enhances its transcriptional activity (PubMed:15890677). Acetylates BCL6 wich disrupts its ability to recruit histone deacetylases and hinders its transcriptional repressor activity (PubMed:12402037). Participates in CLOCK or NPAS2-regulated rhythmic gene transcription; exhibits a circadian association with CLOCK or NPAS2, correlating with increase in PER1/2 mRNA and histone H3 acetylation on the PER1/2 promoter (PubMed:14645221). Acetylates MTA1 at 'Lys-626' which is essential for its transcriptional coactivator activity (PubMed:16617102). Acetylates XBP1 isoform 2; acetylation increases protein stability of XBP1 isoform 2 and enhances its transcriptional activity (PubMed:20955178). Acetylates PCNA; acetylation promotes removal of chromatin-bound PCNA and its degradation during nucleotide excision repair (NER) (PubMed:24939902). Acetylates MEF2D (PubMed:21030595). Acetylates and stabilizes ZBTB7B protein by antagonizing ubiquitin conjugation and degragation, this mechanism may be involved in CD4/CD8 lineage differentiation (PubMed:20810990). Acetylates GABPB1, impairing GABPB1 heterotetramerization and activity (By similarity). In addition to protein acetyltransferase, can use different acyl-CoA substrates, such as (2E)-butenoyl-CoA (crotonyl-CoA), butanoyl-CoA (butyryl-CoA), 2-hydroxyisobutanoyl-CoA (2-hydroxyisobutyryl-CoA) or propanoyl-CoA (propionyl-CoA), and is able to mediate protein crotonylation, butyrylation, 2-hydroxyisobutyrylation or propionylation, respectively (PubMed:17267393, PubMed:25818647, PubMed:29775581). Acts as a histone crotonyltransferase; crotonylation marks active promoters and enhancers and confers resistance to transcriptional repressors (PubMed:25818647). Histone crotonyltransferase activity is dependent on the concentration of (2E)-butenoyl-CoA (crotonyl-CoA) substrate and such activity is weak when (2E)-butenoyl-CoA (crotonyl-CoA) concentration is low (PubMed:25818647). Also acts as a histone butyryltransferase; butyrylation marks active promoters (PubMed:17267393). Acts as a protein-lysine 2-hydroxyisobutyryltransferase; regulates glycolysis by mediating 2-hydroxyisobutyrylation of glycolytic enzymes (PubMed:29775581). Functions as a transcriptional coactivator for SMAD4 in the TGF-beta signaling pathway (PubMed:25514493). Acetylates PCK1 and promotes PCK1 anaplerotic activity (PubMed:30193097). Acetylates RXRA and RXRG (PubMed:17761950).
- 标签蛋白介绍: 粒曼生物定点Knock-in 体系能特异剪切人类第19 号染色体上的 AAVS1 位点，生成DNA 双链断裂（DSB），触发DNA 的自然修复机制，诱导位点与 AAVS1 供体 DNA 克隆之间发生同源重组（HR），将供体克隆上的 DNA 片段整合到基因组上的 safe harbor 位点。HiBiT小分子生物发光标签，分子大小1.3 kDa（11 个氨基酸残基）凭借 “尺寸极小、检测灵敏、无背景干扰” 的核心优势，成为蛋白表达追踪、互作分析、活细胞成像等研究的理想工具，尤其适合对标签干扰敏感的实验场景.
- 细胞背景: [HEK-293]细胞是剪切过的人腺病毒5（Ad5）转染的人胚肾细胞形成的永生化细胞，293 [HEK-293]细胞包含并表达转染的Ad5基因。早期报道中指出，293 [HEK-293]细胞基因组中含有腺病毒5（Ad5）基因组的左侧端和右侧端的DNA，但是现在明确了只存在其左侧端的DNA。经过对Ad5的插入点的克隆测序发现，Ad5的1-4344位线性核苷酸整合入293 [HEK-293]细胞19号染色体（19q13.2）。293 [HEK-293]细胞为人类腺病毒载体扩增的宿主，可表达异常的玻连蛋白的细胞表面受体，由整合素β1亚单位和玻连蛋白受体α-v亚单位组成。
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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：1/3 到 1/5 传代，2-3 天长满。 -
01. 待培养瓶中细胞汇合度至 80%-90% 以上，可进行细胞传代。
02. 将培养基、PBS、胰酶（0.25%Trypsin_EDTA Gibco 25200-056） 等从 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. 为避免冲散细胞，沿培养瓶上壁 PBS 润洗细胞，清洗细胞后弃去，T25 加 2mL。
05. 加入对应体积的胰酶（T75 加 1.5mL， T25 加 0.5mL） ，并轻轻晃动瓶身使胰酶平铺满细胞 底部。可根据实际情况适当增加或减少用量。约 1-2min 后大部分细胞脱落时，加入对应体积的完全培养基终止消化，并用 5mL 移液管轻轻吹打至细胞全部脱落。
06. 将细胞悬液转移至 15mL 离心管，悬液 300g 离心 5min，弃上清。
07. 移取 5mL 完全培养基重悬细胞，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，加 1% 双抗。
08. 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. 对细胞进行消化及离心处理（具体步骤参考传代培养流程）
04. 按照每管 1mL 的量添加冻存液重悬细胞，吹打均匀后分装至冻存管。
05. 将细胞放在程序降温盒中，在 -80℃冰箱中冷冻。
06. 后续将细胞转移到液氮罐中，以便长期储存。 - 粒曼检测HiBiT表达使用The Nano-Glo® HiBiT Lytic Detection System，该系统使用简单的加样-混合-读数检测操作流程，灵敏地定量细胞裂解物中的HiBiT标签蛋白。
产品类型： HiBiT报告细胞系
| 阳性对照试剂盒（TRAC_gene） | 定制靶基因敲除试剂盒-基础版 | 定制靶基因敲除试剂盒-加强版 | |
| 试剂组成： | |||
| sgRNA引物 | √ | √ | √ |
| PCR引物 | √ | √ | √ |
| 测序引物 | √ | √ | √ |
| 细胞裂解液 | √ | √ | √ |
| cas9蛋白 | √ | × | √ |
| 转染试剂 | √ | × | √ |
| 产品规格 | 3次反应 | 5-10次反应 | 5-10次反应 |
| 交付周期 | 1-2周 | 1-2周 | 1-2周 |
| 交付标准 | 经验证，敲除效率最高可达100%，平均敲除效率90% | 经验证，敲除效率在70%以上(通过对照试剂盒优化细胞转染条件后保障效率) | 经验证，敲除效率在70%以上(通 过对照试剂盒优化细胞转染条件后 保障效率) |
| 价格 | RMB：899 | RMB：1899 | RMB：2699 |
基因敲除试剂盒信息
Gene Symbol
NCBI Gene ID
Ensembl ID
Uniprot ID
规格
物种
储存条件
试剂组成
基因敲除试剂盒说明
产品优势
应用