This is a review about phosphotyrosine antibodies (phospho-tyrosine antibodies), based on 77 published articles as of February 15, 2013, using phosphotyrosine antibodies in western blot, immunoprecipitation, immunocytochemistry, and other immunological technologies in randomly selected publications that Labome surveyed systematically. Labome is continuously curating antibodies in formal publications. A summary of phosphotyrosine antibody with an updated list of articles, can be seen here. There are numerous additional publications using phosphotyrosine antibodies. This review is aimed to facilitate Labome visitors in locating the best-suited phosphotyrosine antibodies. Phosphotyrosine antibodies from different suppliers are linked below or can be searched here. The article also addresses some of commonly asked questions about phosphotyrosine antibodies.

Tyrosine phosphorylation is one of the major means of cell signaling transduction and enzymatic activity regulation. The advent of anti-phospho-tyrosine antibodies is one of the significant events in signal transduction research. Before the availability of anti-phosphotyrosine antibodies, tyrosyl phosphorylation of proteins and enzymes was investigated through hazardous and time-consuming radioactive experiments [1]. Anti-phosphotyrosine antibodies are commonly used in western blots after the targeted proteins have been immunoprecipitated to measure the tyrosyl phosphorylation of the proteins. Anti-phosphotyrosine antibodies are also directly used on cell lysates to examine the overall change of tyrosine phosphorylation level in response to various treatments.

Figure 1. An example of phosphorylation studies before the usage of anti-phosphotyrosine antibodies. From [1].

Review of other general antibodies: tag antibodies ( HA, c-Myc and His6); western blot loading controls ( beta actin, alpha tubulin); secondary antibodies.

Table 1 lists the suppliers of phosphotyrosine antibodies among the publications surveyed by Labome.

Among the 22 articles which indicated the clone names, 17 articles listed 4G10, and 6 articles listed PY20 clone (one article listed both clones [2] ) .

Table 2 lists the major applications of phosphotyrosine antibodies among the publications.

EMD Millipore phosphotyrosine antibody

Upstate Biotechnology seems to have been providing some of most commonly used anti-phospho-tyrosine antibodies, including the most commonly used clone 4G10. More than half of the publications cited Upstate anti-phospho-tyrosine antibodies in western blot, immunoprecipitation, and immunocytochemistry.

Most recently, Upstate anti-phosphotyrosine antibody was used to perform western blot and immunohistochemistry in order to study the function of sema3E-plexin D1 [3] , to perform immunoprecipitation in order to study the roles of LRP1 in the adult CNS [4] , and western blot analysis (clone 4G10) in order to determine the receptor property of erythropoietin receptor during receptor signaling activation [5] and to study the effect of the facilitation of AMPA receptor synaptic delivery on cognitive enhancement [6].

Among papers published earlier in 2001, K Aoyama used 4G10 in western blot to study a novel dual specificity phosphatase, LMW-DSP2; K Sauer et al. used 4G10 in western blot to examine the interaction of HPK1 with BLNK in B cells and with SLP-76 in T cells, A E Aplin et al. used 4G10 in western blot to study the regulation of ERK nuclear translocation and Elk-1 phosphorylation by integrin-mediated adhesion; H Konishi et al. used it in western blot to study the tyrosine phosphorylation sites of PKC delta in the H2O2-treated cells; C C Chen used both clone PY-20 and clone 4G10 in western blot and immunocytochemistry to study the induction of adhesive signaling in primary human skin fibroblasts; C Gilbert used 4G10 in immunoprecipitation to evaluate the SAM68 roles in signal transduction during human neutrophil phagocytosis.

In 2002, clone 4G10 was used in western blots by Matthew N Poy to study the mitogenic action of insulin regulated by Shc and CEACAM1 interaction, by Kenneth L Byron to examine vasopressin signal transduction in A7r5 vascular smooth muscle cells, by Diane Lejeune et al. to study STAT3 serine phosphorylation, and in immunocytochemistry by Ann L Moor to localize tyrosine-phosphorylated PLC (pY-PLC) in HTC cells.

In 2003, Yosuke Minami et al. conducted western blot experiments with Upstate Biotechnology antiphosphotyrosine antibody to differentiate the antiapoptotic pathways between wild-type and mutated FLT3; K C Leung et al. used 4G10 in western blot to study the inhibiting effects of estrogen on transcriptional action of GH through the SOCS-2 mediated JAK/STAT pathway; John H Griffin used 4G10 in western blot to study a fusion kinase in EOL-1 cells and the idiopathic hypereosinophilic syndrome.

Matthew L Jones in 2004 used 4G10 to immunoprecipitate phosphorylated SHP-1 proteins; Tod A Brown used anti-phosphotyrosine monoclonal antibody in western blot to elucidate the tyrosine phosphorylation of a novel membrane glycoprotein p80/gp140/CUB domain-containing protein 1 in epithelia; Charng-Jui Chen et al. used 4G10 in western blot and immunocytochemistry to investigate the inhibition of IL-2 production and proliferation in human T cells by carcinoembryonic antigen-related cellular adhesion molecule 1.

In 2005, Upstate Biotechnology anti-phosphotyrosine antibodies were used in western blots to examine the Rac-MKK3/6-p38 pathway in breast epithelial cells by Ilchung Chin, the regulation of the endothelial cell response to Ang-1 through stable interaction between alpha5beta1 integrin and Tie2 tyrosine kinase receptor by Ilaria Cascone et al., the direct interaction between human CNK1 and Raf-1 by Algirdas Ziogas et al., and T-cell receptor-induced activation of phospholipase C-gamma1 by a sequence-independent function of the P-I region of SLP-76 by Ronnie Gonen et al.. Upstate Biotech anti-phosphotyrosine antibody was used in immunoprecipitation to study the phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture by Dr. Amanchy in 2005.

Upstate Biotechnology anti-phosphotyrosine(1:3000) was used in western blot to demonstrate that lipid rafts sequester Ret away from the degradation machinery to sustain Ret signaling by Dr. Pierchala in 2006. Upstate/Millipore mouse antiphosphotyrosine (4G10) antibody was used in western blot to study the role of MNAR in 17beta-estradiol (E2)-induced activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by Dr.Greger in 2007. [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [5] [17] [18] [19] [6] [20] [21] [22] [23] [24] [2] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [4] [42] [3].

Santa Cruz Biotechnology phosphotyrosine antibody

Santa Cruz antiphosphotyrosine (anti-pTyr) antibody was used in western blots to study the downregulation of the Ras-mitogen-activated protein kinase pathway by S Elowe in 2001, and to study the role of beta-arrestin in ubiquitination and IGF-1 receptor down-regulation by Leonard Girnita in 2005. Its PY20 anti-phosphotyrosine antibody was used in immunoprecipitation to study the IL-2 production and proliferation in human T cells inhibited by the cell-cell adhesion molecule carcinoembryonic antigen-related cellular adhesion molecule 1 by Charng-Jui Chen 2004. Matthew N Poy in 2002 also used anti-phosphotyrosine antibody (PY20) in immunoprecipitation to study the regulation of insulin mitogenic action by Shc and CEACAM1 interaction.Santa Cruz Biotechnology phosphotyrosine antibody was used in western blot to investigated the mechanism and function of ubiquitination of protease-activated receptor 2 (PAR2) by Dr. Jacob in 2005. [43] [44] [45] [46] [47] [48] [49] [50] [51] [29] [28] [52].

Cell Signaling Technology phosphotyrosine antibody

New England Biolabs mouse antiphosphotyrosine (P-Tyr100; 9411) antibody was used in western blot to study the effects of IFN tau on STAT activation, expression, DNA binding, and transcriptional activation in an ovine endometrial epithelial cell line by D M Stewart in 2001. [53] [54] [55] [56].

Koichi Seta in 2003 used Transduction Laboratories horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody (RC20H) in western blot to study the angiotensin II-induced trans-activation of the EGF receptor and found that the transactivation was mediated by phosphorylation of tyrosine 319 of the angiotensin II type 1 receptor. The same clone was used by C Sweeney in 2001 in western blot to examine ErbB receptor-mediated signaling pathway stimulation and gene expression. Wen Hong Shen et al.in 2004 used a different Transduction Laboratories mouse anti-phosphotyrosine clone (PY20) in both western blot and immunoprecipitation to study cyclin A expression and retinoblastoma hyperphosphorylation. In addition, Transduction Laboratories polyclonal anti-phosphotyrosine antibody was used in immunoprecipitation to study vasopressin-induced signal transduction in A7r5 vascular smooth muscle cells by Kenneth L Byron in 2002. Transduction Laboratories peroxidase-conjugated anti-phosphotyrosine antibody was used in western blots to exploit EphA2 as a therapeutic target of cancer by Dr. Koolpe in 2002. [57] [58] [59] [60] [61] [8] [62] [63] [64] [65] [66] [26].

In 2001, L You used Sigma monoclonal phosphotyrosine antibody in western blot to study migration and signal transduction induced by cell-derived neurotrophic factor (GDNF) in corneal epithelial cells [67]. In separate experiments, Ret and paxillin proteins were immunoprecipated by their respective antibodies, and their tyrosyl phosphorylation was evaluated in Western blot using Sigma phosphotyrosine antibody as the probe.

Amersham Biosciences monoclonal anti-phosphotyrosine horseradish peroxidase-conjugated antibody was used in western blot to study insulin receptor catalytic activity inhibition by in 2002 [68].

Yutaka Masuda in 2004 used ICN Biomedicals monoclonal anti-phosphotyrosine antibody PY20 in western blot to study the involvement of tumor necrosis factor receptor-associated protein 1 (TRAP1) in beta-hydroxyisovalerylshikon-induced apoptosis. [69] [70].

There are several ways.

• use a phosphorylation-specific antibody against the specific protein.
• use an antibody against the specific protein to immunoprecipitate both phosphorylated or non-phosphorylated forms of the specific protein, then detect with phosphotyrosine antibodies in Western blot.
• use phosphotyrosine antibodies to immunoprecipitate all tyrosine-phosphorylated proteins, and then detect with an antibody against the specific protein.
• mass spec.

BSA should be used, since nonfat dry milk contains proteins that are phosphorylated at tyrosine residues.

the samples should generally be used fresh, since the proteins might be degraded, and dephosphorylation may occur due to phosphatases.

we do not know the answer to this question. Anti-phosphotyrosine anybodies have been used for Western blot, immunoprecipitation, immunocytochemistry, and immunohistochemistry.