| Product Type | Assay Kit |
| Units | 5000 Tests |
| Application | ELISA |
BackgroundThe BrdU Chemiluminescent Cell Proliferation Assay Kit is a non-isotopic enzyme immunoassay for the quantification of DNA synthesis and cell proliferation. Evaluation of cell cycle progression is essential for investigations in many scientific fields. Measurement of [3H] thymidine incorporation as cells enter S phase has long been the traditional method for the detection of cell proliferation. Subsequent quantification of [3H] thymidine is performed by scintillation counting or autoradiography. This technology is slow, labor intensive and has several limitations including the handling and disposal of radioisotopes and the necessity of expensive equipment. A well-established alternative to [3H] thymidine uptake has been demonstrated by numerous investigators. In these methods bromodeoxyuridine (BrdU), a thymidine analog, replaces [3H] thymidine. BrdU is incorporated into newly synthesized DNA strands of actively proliferating cells. Following partial denaturation of double stranded DNA, BrdU is detected immunochemically allowing the assessment of the population of cells which are actively synthesizing DNA. Exalpha Biologicals BrdU Chemiluminescent Cell Proliferation Assay Kit involves incorporation of BrdU Reagent into cells cultured in microtiter plates using the cell layer as the solid phase. The resultant assay is rapid, easy to perform and applicable to high sample throughput. In addition to evaluation of cell proliferation, information such as cell number, morphology and analysis of cellular antigens can be obtained from a single culture.
ApplicationsOptimal concentration should be evaluated by serial dilutions.
StorageExalpha’s BrdU Chemiluminescent Cell Proliferation Assay Kit components are shipped on cold pack. Upon receipt, store kit at -20°C in a non-frost- free freezer. For long term storage, it is recommended that you aliquot and freeze the Prediluted Anti-BrdU Detector Antibody (Component 3) and 2000x Peroxidase Goat anti-Mouse IgG (Component 5) at -20°C. Thirty (30) minutes prior to the use of each component, thaw component. Remove the Fixative/Denaturing Solution (Component 2) and place at room temperature for at least 4 hours prior to use. The Fixative/Denaturing Solution may contain slight precipitation and its color may vary between clear to light yellow.Return the Prediluted Anti-BrdU Detector Antibody (Component 3) and 2000x Peroxidase Goat anti-Mouse IgG (Component 5) to -20°C immediately after use. All other components may be stored at 4-8°C immediately after use. Special care should be taken to keep the Prediluted AntiBrdU Detector Antibody (Component 3) and 2000x Peroxidase Goat anti-Mouse IgG (Component 5) cold by pulling out the number of aliquots needed for the test, keeping them on ice, and leaving the remaining aliquots at -20°C.
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.
Kit ManualClick here to download the Kit Manual Safety Datasheet(s) for this product:NM_X1623K SDS_V1/wp-content/uploads/SDS/X1623K SDS_V1.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.

