| Clone | AM1 4 |
| Isotype | IgG |
| Product Type | Monoclonal Antibody |
| Units | 100 µg |
| Host | Mouse |
| Species reactivity | Human |
| Application | Western Blotting |
BackgroundCaspase-3 along with caspase 7 and 6 form the group of effector caspases that are responsible for the cleavage of multiple substrates including the cytokeratins, PARP, alpha fodrin, NuMA and others.Caspase-7 occurs in three varient forms.Caspase-3-like activities are required for Fas-mediated apoptosis. However, the role of caspase-1 and caspase-3 in mediating Fas-induced cell death is not clear. Although wild-type, caspase-1(-/-), and caspase-3(-/-) hepatocytes were killed at a similar rate when cocultured with FasL expressing NIH 3T3 cells, caspase-3(-/-) hepatocytes displayed drastically different morphological changes as well as significantly delayed DNA fragmentation. For both wild-type and caspase-1 (-/-) apoptotic hepatocytes, typical apoptotic features such as cytoplasmic blebbing and nuclear fragmentation are seen within 6 hr, but neither event was observed for caspase-3(-/-) hepatocytes. In thymocytes apoptotic caspase-3 (-/-) thymocytes exhibit similar abnormal morphological changes and delayed DNA fragmentation observed in hepatocytes. Cleavage of various caspase substrates implicates apoptotic events, including gelsolin, fodrin, laminB, and DFF45/ICAD are delayed or absent. The altered cleavage of these key substrates is likely responsible for the aberrant apoptosis observed in both hepatocytes and thymocytes deficient in caspase-3.
Synonyms: Cysteine-requiring Aspartate Protease-3
Source
Immunogen: Hybridoma produced by the fusion of splenocytes from mice immunized with recombinant human Caspase-3 protein and mouse myeloma cells.
Product
Product Form: Unconjugated
Formulation: Provided as solution in phosphate buffered saline with 0.08% sodium azide
Purification Method: Protein A/G Chromatography
Concentration: See vial for concentration
ApplicationsDetects human Caspase-3 by Western blot.Optimal concentration should be evaluated by serial dilutions.
Functional Analysis: Western Blotting
StorageProduct should be stored at -20ºC. Aliquot to avoid freeze/thaw cycles
Product Stability: Products are stable for one year from purchase when stored properly
Shipping Conditions: Ship at ambient temperature, freeze upon arrival
CautionThis product is intended FOR RESEARCH USE ONLY, and FOR TESTS IN VITRO, not for use in diagnostic or therapeutic procedures involving humans or animals. It may contain hazardous ingredients. Please refer to the Safety Data Sheets (SDS) for additional information and proper handling procedures. Dispose product remainders according to local regulations.This datasheet is as accurate as reasonably achievable, but Nordic-MUbio accepts no liability for any inaccuracies or omissions in this information.
References1. Slee, E.A., et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J. Cell Biol. 1999, 144, 281-2922. Ueda, S., et al. Redox regulation of caspase-3(-like) protease activity: regulatory roles of thioredoxin and cytochrome c. J. Immunol. 1998, 161, 6689-6695 3. Samali, A., et al. Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells. EMBO J. 1999, 18, 2040-20484. Cohen, G.M., et al. Caspases: the executioners of apoptosis. Biochem. J. 1997, 326, 1-16
Protein Reference(s)
Database Name: SwissProt
Accession number: P42574
Species Accession: Human
Safety Datasheet(s) for this product:NM_Sodium Azide/wp-content/uploads/SDS/Antibody SDS with Sodium Azide Noridic-MUbio.pdf
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其次,察看次目的蛋白的存在形式,有没有多聚体形式及变构形式;
最后,查看多家抗体公司的DATA,看看别人的WB做出来的条带的位置。
根据你说的,特异识别多个组织中的同样大小的条带,我觉得很可能就是你的目的蛋白。
一般来说多克隆的阳性率高一些,但出现假阳性的比例也高一些。
1取组织
(1) 灌注固定:暴露心脏,将注射针头插入左心室,剪开右心耳,快速灌注PBS,待右心耳处流出澄清液体,换4%多聚甲醛灌注到大鼠四肢强直为止,快速取脑和肾。
(2) 固定:将组织置于4%多聚甲醛中固定15小时
(3) 脱水:将组织放入30% 蔗糖溶液中直至下沉
(4) 切片:脑组织冰冻切片20微米,37度烘干2h以上;
(5)将切片放置-80度保存,随用随取。
2免疫单标记
(1)做抗原修复(0.4g柠檬酸、3g柠檬酸钠、1000ml水配制成溶液,放到水浴箱中90~94℃保存15min),冷却后, 用1*PBS反复洗标本10min,吸去PBS,同时向标本加渗透液(配0.4%trition液,然后将BSA加入到配好的trition液中,配成1%浓度)100ul,常温下湿盒反应1h。
(2) 吸去渗透液,同时向标本加block液(5%封闭用正常山羊血清)100ul,常温下湿盒反应5h。
(3) 吸去block液,同时向标本加入1:50稀释的羊抗鼠一抗100ul,37度湿盒反应过夜。
(4)吸去一抗,并回收,用1*PBS洗液反复洗15min。
(5)快速从冰箱中取出驴抗羊的二抗,并立即用锡纸包裹,注入到一个新的以锡纸包裹的tube管中并用PBS以1:200稀释。
(6)进入暗室,吸去PBS,同时加入二抗100ul,37度湿盒反应2h,封片。
(7)放置到荧光显微镜下观察。
又由于自然存在的抗原大都存在多个抗原表位,会刺激机体产生多种针对同一抗原的不同抗原表位相应的不同抗体.

