The specific binding of phosphoinositides by proteins is a main determinant in localizing those proteins to their site of function in various cellular processes, such as actin cytoskeletal organization, control of gene expression, cell growth regulation, exocytosis and endocytosis. The most understood phosphoinositide-binding motifs are the C2 (PKC conserved region 2), PH (Pleckstrin homology), FYVE (Fab1p/YOTP/Vac1p/EEA1), ENTH (Epsin NH2-terminal homology) and PX (Phox homology) domains.
Echelon Biosciences has developed PIP Arrays and Strips to facilitate the analysis of phosphoinositide protein interactions by protein¡Vlipid overlay assays. PIP strips allow users to measure the relative affinity of a certain protein for a set of phosphorylated lipids. The phosphoinositide (PIP) family consists of eight specific phospholipids with different head groups. Echelon's PIP Array consists of seven concentrations of the eight PIPs noncovalently adsorbed to a nitrocellulose membrane. The PIP Strips contain the following lipids distributed in a specific layout on the membrane: lysophosphatidic acid [LPA], Lysophosphocholine [LPC], Phosphatidylinositol [PtdIns, PI], Phosphatidylinositol (3) phosphate [PtdIns(3)P, PI(3)P] Phosphatidylinositol (4) phosphate [PtdIns(4)P, PI(4)P], Phosphatidylinositol (5) phosphate [PtdIns(5)P, PI(5)P], Phosphatidylethanolamine [PE], Phosphatidylcholine, [PC], Sphingosine-1-Phosphate, [S1P], Phosphatidylinositol (3,4) bisphosphate [PtdIns (3,4)P, PI(3,4)P2], Phosphatidylinositol (3,5) phosphate [PtdIns(3,5)P, PI(3,5)P2], Phosphatidylinositol (4,5) bisphosphate [PtdIns(4,5)P, PI(4,5)P2], Phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P, PI(3,4,5)P3], phosphatidic acid [PA], Phosphatidylserine [PS]. PIP Strips contain 100 picomoles per spot of all eight phosphoinositides and other biologically active lipids. It is recommended to store them at 2-8¢XC in a dry place.
PIP Strips replace time-consuming binding assays and the technique requires only 0.5 ug of protein. A positive control of GST-tagged lipid recognition protein G-1100 is available with the strips. The protocol is pretty straightforward and resembles a classic Western blot procedure. First, the membrane is incubated with a blocking agent with gentle agitation for 1 h at room temperature. One can use either TBS-T, PBS-T, TBS-T or PBS-T with 3% BSA, TBS-T or PBS-T with 1% milk, TBS-T or PBS-T with 0.1% ovalbumin. Then the membrane is incubated with the protein of interest (1 microgram/ml protein in blocking solution) either 1 hour at room temperature or overnight at 4„aC. The membrane is washed 3 times for 10 minutes then the anti-GST antibody is added and the membrane is incubated with it for 1 hour at room temperature. I use the mouse monoclonal anti-GST antibody from Cell Signaling Technologies diluted 1:3000 in blocking solution. Next, the membrane is washed as above and then incubated with anti-mouse horseradish peroxidase (HRP) antibody diluted 1:3000 in blocking solution. The secondary antibody HRP conjugate should not be used in the presence of sodium azide or hemoglobin and during all incubations, great care must be taken not to let the membrane dry. After incubation with the secondary, the membrane is washed as before then incubated with ECL detection solution from GE Healthcare. The membrane is exposed to film and then the signal is detected using an imager for chemiluminescent detection. All incubation times and concentrations should be optimized for each specific protein.
Results are easy to obtain, but they should be compared with results from alternative assays, such as PolyPIposomes, PIP Beads and PIP plates, also offered by Echelon. When we want to verify the biological relevance of PIP strips results, we use PIP beads. PIP beads are designed for use in protein pull-down experiments to identify and characterize phosphoinositide binding proteins from cell lysates or from a mixture of in vitro translated peptides. The interacting proteins are further analyzed by SDS-PAGE electrophoresis. I successfully used PIP Strips to define the lipid interactions of 2 Drosophila protein fragments, a C terminal fragment of anillin and of a central domain of septin 2.
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