抗体的生物素化标记实验要点：

1. 磷酸化酪氨酸激酶B抗体规格 如在反应混合液中有叠氮钠或游离氨基存在,会抑制标记反应。因此,蛋白质在反应前要对 0.1mol/L碳酸氢钠缓冲液或0.5mol/L硼酸缓冲液充分透析；

2.所用的NHSB及待生物素化蛋白质之间的分子比按蛋白质表面的ε-氨基的密度会有所不同,选择不当则影响标记的效率,应先用几个不同的分子比来筛选最适条件；

3.用NHSB量过量也是不利的,抗原的结合位点可能因此被封闭,导致抗体失活；

4.由于抗体的氨基不易接近可能造成生物素化不足,此时可加入去污剂如 Triton x-100, Tween20等；

5.当游离ε-氨基(赖氨酸残基的氨基)存在于抗体的抗原结合位点时,或位于酶的催化位点时,生物素化会降低或损伤抗体蛋白的结合力或活性；

6.生物素还可能与不同的功能基团,如羰基、氨基、巯基、异咪唑基及*基,也可与糖基共价结合；

7.交联反应后,应充分透析,否则,残余的生物素会对生物素化抗体与亲和素的结合产生竞争作用；

8.在细胞的荧光标记实验中,中和亲和素的本底低,但由于链霉亲和素含有少量正电荷,故对某些细胞可导致高本底。

产品订购信息：
英文名称  Anti-phospho-TrkB (Tyr705) 

中文名称   磷酸化酪氨酸激酶B抗体规格 

别    名  TrkB (Phospho-Tyr705); phospho-NTRK2(Tyr705); NTRK2(phospho Y705); TrkB; Tyrosine Receptor Kinase B; Tyrosine kinase,Pyk2; BDNF tropomyosine receptor kinase B; BDNF/NT 3 growth factors receptor; BDNF/NT-3 growth factors receptor; Brain derived neurotrophic factor receptor; GP145 TrkB; GP145-TrkB; GP145-TrkB/GP95-TrkB; GP95 TrkB; Neurotrophic tyrosine kinase receptor type 2; Neurotrophin receptor tyrosine kinase type 2; NTRK 2; Ntrk2; NTRK2_HUMAN; Obesity, hyperphagia, and developmental delay, included; RATTRKB1; Tkrb; Trk B; Trk-B; TRKB; TrkB tyrosine kinase; TRKB1; tyrosine kinase receptor B; Tyrosine receptor kinase B.

浓    度   1mg/1ml

规 格  0.1ml/100μg

抗体来源   Rabbit

克隆类型   polyclonal

交叉反应   Human, Mouse, Rat, Chicken, Dog, Pig, Cow, Horse, Rabbit 

产品类型   一抗 磷酸化抗体  

研究领域    免疫学 神经生物学 信号转导 转录调节因子 激酶和磷酸酶

蛋白分子量  predicted molecular weight: 92kDa

性    状   Lyophilized or Liquid

免 疫 原 KLH conjugated Synthesised phosphopeptide derived from rat NTRK2 around the phosphorylation site of Tyr705 

亚    型   IgG

纯化方法   affinity purified by Protein A

储 存 液   0.01M PBS, pH 7.4 with 10 mg/ml BSA and 0.1% Sodium azide

磷酸化酪氨酸激酶B抗体规格 产品应用    WB=1:100-500 IP=1:20-100 IHC-P=1:100-500 IHC-F=1:100-500 IF=1:100-500

（石蜡切片需做抗原修复） 

 not yet tested in other applications.

 optimal dilutions/concentrations should be determined by the end user.  

保存条件  Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibody is stable at room temperature for at least one month and for greater than a year when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C. 

Important Note  This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications. 

产品介绍 The Trk family of nerve growth factor receptors includes Trk A(also referfed to as Trk A gp140),Trk B and Trk C. The prototype member of this gene family, Trk A, encodes a 140 kDa cell surface receptor, gp140, the expression of which is restricted in vivo to neurons of the sensory spinal and cranial gangliaof neurocrest origin. Nerve growth factor (NGF) stimulates tyrosine phosphorylation of Trk gp 140 in neural cell lines and in embryonic dorsal root ganglia. By comparison, BDNF and to a lesser extent, NT-3, but not NGF, can induce tyrosine phophorylayion of Trk B gp 145. The third member of the Trk receptor family, Trk C incodes a 140 kDa protein, Trk C gp140, that is preferentially expressed in brain tissue and primarily functions as a receptor for NT-3.An additional component of the Trk receptor complex, NGFR p175, binds to neurotrophic factors with low affinity but is required for efficient signaling. NGFR p175 accelerates Trk activation and may recruit downstream dffector molecules to the ligand-bound receptor complex.

Function : Receptor tyrosine kinase involved in the development and the maturation of the central and the peripheral nervous systems through regulation of neuron survival, proliferation, migration, differentiation, and synapse formation and plasticity. Receptor for BDNF/brain-derived neurotrophic factor and NTF4/neurotrophin-4. Alternatively can also bind NTF3/neurotrophin-3 which is less efficient in activating the receptor but regulates neuron survival through NTRK2. Upon ligand-binding, undergoes homodimerization, autophosphorylation and activation. Recruits, phosphorylates and/or activates several downstream effectors including SHC1, FRS2, SH2B1, SH2B2 and PLCG1 that regulate distinct overlapping signaling cascades. Through SHC1, FRS2, SH2B1, SH2B2 activates the GRB2-Ras-MAPK cascade that regulates for instance neuronal differentiation including neurite outgrowth. Through the same effectors controls the Ras-PI3 kinase-AKT1 signaling cascade that mainly regulates growth and survival. Through PLCG1 and the downstream protein kinase C-regulated pathways controls synaptic plasticity. Thereby, plays a role in learning and memory by regulating both short term synaptic function and long-term potentiation. PLCG1 also leads to NF-Kappa-B activation and the transcription of genes involved in cell survival. Hence, it is able to suppress anoikis, the apoptosis resulting from loss of cell-matrix interactions. May also play a role in neutrophin-dependent calcium signaling in glial cells and mediate communication between neurons and glia.Subunit : Exists in a dynamic equilibrium between monomeric (low affinity) and dimeric (high affinity) structures. Interacts (phosphorylated upon activation by BDNF) with SHC1; mediates SHC1 phosphorylation and activation. Interacts (phosphorylated upon activation by BDNF) with PLCG1 and/or PLCG2; mediates PLCG1 phosphorylation and activation. Interacts with SH2B1 and SH2B2. Interacts with NGFR; may regulate the ligand specificity of the receptor. Interacts (phosphorylated upon ligand-binding) with SH2D1A; regulates NTRK2. Interacts with SQSTM1 and KIDINS220. Interacts (phosphorylated upon ligand-binding) with FRS2; activates the MAPK signaling pathway.Subcellular Location : Cell membrane; Single-pass type I membrane protein. Endosome membrane; Single-pass type I membrane protein. Note=Internalized to endosomes upon ligand-binding.Tissue Specificity : Isoform TrkB is expressed in the central and peripheral nervous system. In the central nervous system (CNS), expression is observed in the cerebral cortex, hippocampus, thalamus, choroid plexus, granular layer of the cerebellum, brain stem, and spinal cord. In the peripheral nervous system, it is expressed in many cranial ganglia, the ophtalmic nerve, the vestibular system, multiple facial structures, the submaxillary glands, and dorsal root ganglia. Isoform TrkB-T1 is mainly expressed in the brain but also detected in other tissues including pancreas, kidney and heart. Isoform TrkB-T-Shc is predominantly expressed in the brain.Post-translational modifications : Phosphorylated. Undergoes ligand-mediated autophosphorylation that is required for interaction with SHC1 and PLCG1 and other downstream effectors. Isoform TrkB-T-Shc is not phosphorylated.Ubiquitinated. Undergoes polyubiquitination upon activation; regulated by NGFR. Ubiquitination regulates the internalization of the receptor (By similarity).DISEASE : Defects in NTRK2 are the cause of obesity hyperphagia and developmental delay (OHPDD) [MIM:613886]. OHPDD is a disorder characterized by early-onset obesity, hyperphagia, and severe developmental delay in motor function, speech, and language.Similarity : Belongs to the protein kinase superfamily. Tyr protein kinase family. Insulin receptor subfamily.Contains 2 Ig-like C2-type (immunoglobulin-like) domains.Contains 2 LRR (leucine-rich) repeats.Contains 1 LRRCT domain.Contains 1 LRRNT domain.Contains 1 protein kinase domain.Database links : UniProtKB/Swiss-Prot: Q16620.1NGF, BDNF, NT-3, NT-4等的受体包括trkA, trkB, trkC及NGFR。BDNF, NT-3能诱导trkB的酪氨酸磷酸化。(神经生长因子受体的一种)

抗体的鉴定：

1）磷酸化酪氨酸激酶B抗体规格 抗体的效价鉴定：不管是用于诊断还是用于,制备抗体的目的都是要求较高效价。不同的抗原制备的抗体,要求的效价不一。鉴定效价的方法很多,包括有试管凝集反应,琼脂扩散试验,酶联免疫吸附试验等。常用的抗原所制备的抗体一般都有约成的鉴定效价的方法,以资比较。如制备抗抗体的效价,一般就采用琼脂扩散试验来鉴定。

2）抗体的特异性鉴定：抗体的特异性是指与相应抗原或近似抗原物质的识别能力。抗体的特异性高,它的识别能力就强。衡量特异性通常以交叉反应率来表示。交叉反应率可用竞争抑制试验测定。以不同浓度抗原和近似抗原分别做竞争抑制曲线,计算各自的结合率,求出各自在IC50时的浓度,并按公式计算交叉反应率。 

如果所用抗原浓度IC50浓度为pg/管,而一些近似抗原物质的IC50浓度几乎是无穷大时,表示这一抗血清与其他抗原物质的交叉反应率近似为0,即该血清的特异性较好。

3）抗体亲和力：是指抗体和抗原结合的牢固程度。亲和力的高低是由抗原分子的大小,抗体分子的结合位点与抗原决定簇之间立体构型的合适度决定的。有助于维持抗原抗体复合物稳定的分子间力有氢键,疏水键,侧链相反电荷基因的库仑力,范德华力和空间斥力。亲和力常以亲和常数K表示,K的单位是L/mol。抗体亲和力的测定对抗体的筛选,确定抗体的用途,验证抗体的均一性等均有重要意义。

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