Phospho-INAD

Accession # and alternate nomenclature: NP_726260

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. Approximately 120 mb euchromatic portion of the fly genome ha been sequenced using a shotgun sequencing approach. The entire fly genome encodes for approximately 13,600 genes with vast functional diversity. Inactivation-No-activation potential D (INAD) is a scaffolding protein containing five PSD95/dlg/zonular occludens-1 (PDZ) domains that anchor and tether the NORA (Phospholipase C beta), calcium ion channels of the transient receptor potential (TRP) and eye-PKC in drosophila photoreceptor. The second PDZ domain in INAD interacts with eye-PKC via PDZ ligand (Ile, Thr, Ile, Ile) at the carboxy terminus. The coding sequence of INAD contains 9 exons spanning 3kb conceptual translation of the coding sequences resulted in multiple SNPs affecting several residues in the 2 PDZ domain of the INAD protein (1). INAD gene encodes a photoreceptor specific polypeptide of 674 residues, with in this sequence there are 2 repeats that share significant homology with a family of cytoskeleton-associated protein that is involved in signal transduction. Point mutation in INAD215 in photoreceptor cells exhibited slow calcium influx dependent deactivation of the light-induced current (2).

In the Drosophila visual cascade, the transient receptor potential (TRP) calcium channel, phospholipase Cbeta (no-receptor-potential A), and an eye-specific isoform of protein kinase C (eye-PKC) comprise a multi-molecular signaling complex via their interaction with the scaffold protein INAD. The interaction of PDZ2 domain in INAD with TRP-Na+/Ca+2 channel and C-protein coupled phospholipase C suggest that INAD functions as a regulatory subunit of the TRP Ca-channel to facilitate the regulation of upstream signaling molecules. Upon anchoring eye-specific PKC, TRP calcium channel, he INAD undergo phosphorylation near C-terminal end of the protein. The de-phosphorylation of INAD is partially suppressed by protein phosphatase inhibitor Okadaic acid, microcysteine and protein phosphates inhibitor 2. The phosphorylation data suggest that INAD functions like RACK (Receptor for activated protein kinase C) allowing eye-PKC to phsophorylate INAD and TRP (3).

The Phospho-INAD-selective antibodies were generated against synthetic peptides form C-terminal regions where the protein is phosphorylated at Thr-666. The Phospho-threonine containing INAD peptide was post-synthetically modified to achieve the desired antigenicity before injecting in to rabbits to obtained antibodies. The phospho-INAD antibodies were selected on immobilized phosphor- and non-phospho-antigen based affinity chromatography and the purified IgGs are stabilized in antibody stabilization buffer. The phosphor-INAD antibodies labeled phospho-INAD protein and has no affinity for non-phosphoryalted protein. FabGennix Inc. provides Western blot positive control samples for INAD and Phospho-INAD in ready-to-use buffer for SDS-PAGE and western blotting applications. Phospho-INAD positive control appears as a diffuse band of 83kDa. Limited quantities of antigenic blocking peptides for this antibody are also available (Inquire before placing orders). For a complete listing of all Drosophila related antibodies, visit our website at www.FabGennix.com.

Immunogen:            PINAD-140AP antibodies are developed against conserved antigenic peptide sequences corresponding to aa 659-674, the peptide was covalently modified by incorporating Phospho-Threonine to achieve desired antigenicity before used as antigen.

Concentration:         Antibody PINAD-140AP has a concentration of 0.65-0.74mg/ml in antibody stabilization buffer.

Applications:           ELISA/dot blot: Antibody dilution 1:20,000-1:50,000;. Western blot: Antibody dilution 1:500 in diluObuffer. PC-INAD sample 25ul/lane. The antibody labels a single band of blots are blocked in 5XdiluObuffer for WB using PC-PINAD. IMM: n.d; IHC n.d

Reactivity                The antibodies to Phospho-INAD (PINAD-140AP) labels 81-83kDa In-activation-No-After potential D (INAD) protein in PC-INAD samples. N.D does not necessarily means the antibody in not suitable for that application, investigators using this antibody in applications not listed here can ask for a complimentary sample.

Protocols:                Description and use of this antibody in various applications is provided with the product. Standard protocol for various applications (Western blot; immunoprecipitation and immunohistochemistry) of this antibody can be requested by calling our Technical support line. The recommended dilutions are for reference only and FabGennix Inc. strongly recommends investigators to optimize conditions for use of this product in their laboratories.

Form/Storage:         The antiserum is supplied in antibody stabilization buffer with preservatives. For long-term storage of antibody, store at -20^oC FabGennix Inc. does not recommend storage of very dilute antibody solutions unless they are prepared in specially formulated multi use antibody dilution buffer (Cat # DiluOBuffer). Working solutions of antibodies in DiluOBuffer should be filtered through 0.45um filter after every use for long-term storage.