
Neuropeptide Y (NPY) is a member of a regulatory peptide family and has a marked sequence homology with pancreatic polypeptide (PP) and peptide YY (PYY), which are other members of the family. NPY is widely expressed in the nervous system and has been shown to be differentially expressed in inhibitory interneurons in the hippocampus in degenerative disease, as a powerful vasoconstrictor in the periphery, and increased expression of NPY in the hypothalamus correlates with food intake. In the rat central nervous system, immunohistochemistry has found NPY-like cell bodies in the cortex, caudate-putamen, hypothalamus (arcuate nucleus), hippocampus, anterior westolfactory bulb, nucleus accumbens, amygdaloid complex and periaqueductal grey. NPY-like fibers and terminals are detected in high numbers in the bed nucleus of the stria terminalis, the peri- and paraventricular regions of the hypothalamus and thalamus and in discrete hypothalamic nuclei, particularly the suprachiasmatic nucleus.
Westerns are not recommended for the Neuropeptide Y antibody since the protein is too small to detect in gel.
Photo Description: IHC image of neurons staining for neuropeptide Y in the rat cortex (above) and striatum (below). The tissue was fixed with 4% formaldehyde in phosphate buffer, before being removed and prepared for vibratome sectioning. Floating sections were incubated at RT in 10% goat serum in PBS, before standard IHC procedure. Primary antibody was incubated at 1:5000 for 48 hours, goat anti-rabbit secondary was subsequently added for 1 hour after washing with PBS. Light microscopy staining was achieved with standard biotin-streptavidin/HRP procedure and DAB chromogen. The second image above entitled "C." is NPY interneurons (RED) in mouse striatum, GREEN staining is GFP expressed by medium spiny neurons provided Alipi Naydenov, Neurobiology and Behavior, Stella Lab University of Washington (see review below).
Host: Rabbit
Quantity / Volume: 100 µL
State: Lyophilized Whole Serum
Reacts With: Alpaca (Llama), Bird, Bubalus Bubalis (Water Buffalo), Bullfrog, Cat, Ewe, Ferret, Fish, Frog, Frog (Xenopus Laevis), Ground Squirrel, Guinea Pig, Hamster, Human, Monkey, Mouse, Ovine (Sheep), Pig, Psetta Maxima (Flat Fish), Rat, Sheep, Snake, Tadpole, Toad, Turtle, Zebrafish
Availability: In Stock
Preabsorption Control: Available at Sigma Aldrich, Catalog # N3266
Alternate Names: Pro-neuropeptide Y; Neuropeptide tyrosine; prepro-neuropeptide Y; PYY4, anti-NPY
Gene Symbol: NPYRRID: AB_572253
Database Links:
Entrez Gene: 102414548 Bubalus Bubalis (Water Buffalo) Entrez Gene: 101096968 Cat Entrez Gene: 443508 Ewe Entrez Gene: 101686990 Ferret Entrez Gene: 779985 Frog Entrez Gene: 101976112 Ground Squirrel Entrez Gene: 100379621 Guinea Pig Entrez Gene: 100758436 Hamster Entrez Gene: 4852 Human Entrez Gene: 574114 Monkey Entrez Gene: 109648 Mouse Entrez Gene: 397304 Pig Entrez Gene: 24604 Rat Entrez Gene: 102454441 Turtle Entrez Gene: 30281 Zebrafish
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多抗,稀释度更大,特异性相对较差,容易出现多条带。
兔的单克隆抗体和鼠的单克隆抗体在使用上不会有什么区别。
用来很多抗体,许多时候觉得单抗多抗也未必是理论上那样的。单抗做不好的也有,多抗条带唯一且清晰的也有。
而且很多蛋白的抗体未必有那么多的选择。
抗原有两个基本特性,即抗原性和免疫原性。有抗原性的物质不一定有免疫原性,所以由此引出半抗原和完全全抗原,半抗原必须经过经过一定的改造(偶联蛋白载体BSA,OVA或者HSA等大分子物质)方能成为完全。一般而言完全抗原分子量越大(大于10KDa),结构越复杂引起免疫反应的能力也就越强。
抗体就是能与特异性抗原结合的免疫球蛋白,抗体一般分为多克隆抗体和单克隆抗体,多克隆抗体能与抗原的多个表位结合。本篇主要讲述兔来源的多克隆抗体的生产步骤
多抗一般制备流程:完全抗原的准备→兔子的免疫→ 效价检测和终放→抗体亲和纯化→抗体的浓缩和保存。
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.
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。

