Pi 3 p

Manufactured by Echelon Biosciences

Sourced in United States

PI(3)P is a phosphoinositide lipid that serves as a key regulator of cellular processes. It functions as a signaling molecule and plays a crucial role in various cellular pathways.

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Lab products found in correlation

9 protocols using pi 3 p

Purification of Vacuolar Trafficking Proteins

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Lipids were obtained from Avanti Polar Lipids (Alabaster, AL), except for ergosterol which was from Sigma-Aldrich (St. Louis, MO), PI(3)P was from Echelon Biosciences (Salt Lake City, UT), and the fluorescent lipids (Marina-Blue-DHPE, NBD-DHPE, Rhodamine-DHPE, Oregon-Green-488-DHPE, Texas-Red-DHPE) were from Life Technologies (Carlsbad, CA). Biotinylated R-phycoerythrin was purchased from Life Technologies, Cy5-derivatized streptavidin from KPL (Gaithersburg, MD), and unlabeled streptavidin from Thermo Scientific (Waltham, MA). Sec18p (Haas and Wickner, 1996 (link)), Sec17p (Schwartz and Merz, 2009 (link)), Ypt7p (Zick and Wickner, 2013 (link)), HOPS (Zick and Wickner, 2013 (link)), and vacuolar SNARE proteins (Mima et al., 2008 (link); Schwartz and Merz, 2009 (link); Zucchi and Zick, 2011 (link)) were purified as described. Vti1p and Nyv1p were exchanged into octylglucoside buffer as described (Zucchi and Zick, 2011 (link)). Soluble Nyv1p (sR) was purified as GST-TEVsite-Nyv1(Δtm) as described (Thorngren et al., 2004 (link)), and cleaved with TEV protease prior to use. GST-His6-3Csite-Nyv1p was purified as described (Izawa et al., 2012 (link)).

Zick M, & Wickner W.T. (2014). A distinct tethering step is vital for vacuole membrane fusion. eLife, 3, e03251.

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Purification of Vacuole Fusion Proteins

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Lipids were obtained from Avanti Polar Lipids, except ergosterol was from Sigma–Aldrich (St. Louis, MO), PI(3)P was from Echelon Biosciences (Salt Lake City, UT), and the fluorescent lipids were from Life Technologies (Carlsbad, CA). Sec18p (Haas and Wickner, 1996 (link)), Sec17p (Schwartz and Merz, 2009 (link)), Ypt7p (Zick and Wickner, 2013 (link)), HOPS (Zick and Wickner, 2013 (link)), and vacuolar SNARE proteins (Mima et al., 2008 (link); Schwartz and Merz, 2009 (link); Zucchi and Zick, 2011 (link)) were purified as described. Vti1p and Nyv1p were exchanged into octylglucoside buffer as described (Zucchi and Zick, 2011 (link)).

Zick M., Stroupe C., Orr A., Douville D, & Wickner W.T. (2014). Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion. eLife, 3, e01879.

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Lipid Preparation and Storage Protocol

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The following lipids were purchased and used directly: DOPC, DOPS,
DOGS-NiNTA, RhoDPPE (Avanti), DiD (Invitrogen), and PI(3)P (Echelon
Biosciences). Lipids were dissolved in chloroform, except PI(3)P in 1:2:0.8
CHCl₃:MeOH:H₂O. All were stored at -80 °C.

Murray D.H., Jahnel M., Lauer J., Avellaneda M.J., Brouilly N., Cezanne A., Morales-Navarrete H., Perini E.D., Ferguson C., Lupas A.N., Kalaidzidis Y., Parton R.G., Grill S.W, & Zerial M. (2016). An endosomal tether undergoes an entropic collapse to bring vesicles together. Nature, 537(7618), 107-111.

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Lipid Reconstitution and Characterization

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PI3P was from Echelon (Salt Lake City, Utah), ergosterol from Sigma (St. Louis, MO), fluorescent lipids from Thermo Fisher (Waltham, MA), and other lipids were from Avanti (Alabaster, AL). Biobeads SM2 were from BioRad, Cy5-Streptavidin from SeraCare (Milford, MA), biotinylated phycoerythrin from Invitrogen (Eugene, OR), and underivatized streptavidin from Thermo Fisher. Spectrapor six dialysis tubing (7.5 mm diameter, 25 kDa cutoff) was from Spectrum Labs (Las Vegas, NV). Octyl-b-D-glucopyranoside was purchased from Anatrace (Maumee, OH).

Song H., Torng T.L., Orr A.S., Brunger A.T, & Wickner W.T. (2021). Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering. eLife, 10, e67578.

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Purification of Vacuole Fusion Proteins

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The purification of HOPS, prenylated Ypt7 (Zick and Wickner, 2013 (link)), Vam7, Sec17 (Schwartz and Merz, 2009 (link)), Sec18 (Haas and Wickner, 1996 (link)), and neuraminidase (Zucchi and Zick, 2011 (link)) were as described. Membrane-anchored vacuolar SNAREs (Vam3, Vti1, Nyv1) were isolated (Mima et al., 2008 (link)) and exchanged into octylglucoside buffer (Zucchi and Zick, 2011 (link)). Proteins were frozen in aliquots in liquid nitrogen and stored at −80ºC. Most lipids were obtained from Avanti Polar Lipids (Alabaster, AL), with the exception of ergosterol, which was from Sigma-Aldrich (St. Louis, MO), PI(3)P, which was from Echelon Biosciences (Salt Lake City, UT), and the fluorescent lipids (Marina Blue–1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine [DHPE] and NBD-DHPE), which were from Thermo Fisher Scientific (Waltham, MA). Cy5-derivatized streptavidin was purchased from KPL (now SeraCare Life Sciences, Milford, MA) and biotinylated PhycoE and unlabeled streptavidin from Thermo Fisher Scientific. Protein concentrations were determined by Bio-Rad (Hercules, CA) protein assay, which is based on the Bradford dye-binding method (Bradford, 1976 (link)).

Zick M, & Wickner W. (2016). Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement. Molecular Biology of the Cell, 27(16), 2590-2597.

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Purification and Characterization of Rhodopsin

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The rhodopsin 1D4 antibody was a kind gift from Dr. James F. McGinnis (OUHSC). The amylose affinity resin was obtained from New England Bio labs (Ipswich, MA, USA). All other reagents were of analytical grade and purchased from Sigma (St. Louis, MO, USA). Glutathione agarose beads were obtained from ThermoFisher Scientific (Waltham, MA, USA). PIP strips, PI(3)P, PI(4)P, PI(5)P, PI(3,4)P₂, PI(4,5)P₂ and PI(3,4,5)P₃ lipids, PI3-kinase alpha (PI3Kα) active enzyme, and PTEN enzyme were obtained from Echelon Biosciences (Salt Lake City, UT, USA).

Rajala A., McCauley A., Brush R.S., Nguyen K, & Rajala R.V. (2020). Phosphoinositide Lipids in Ocular Tissues. Biology, 9(6), 125.

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Lipid Reagent Procurement and Handling

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Lipids were purchased from Avanti Polar Lipids (Alabaster, AL) with the exceptions of PI₃P (p hosphatidylinositol 3-phosphate diC16) from Echelon Biosciences (Salt Lake City, UT) and ergosterol from Fluka (St. Louis, MO). Octylglucoside (n-octyl-ß-d-glucopyranoside) was from Anatrace (Maumee, OH), Cy5-labeled streptavidin from LGC Clinical Diagnostics (Milford, MA), biotin-conjugated R-phycoerythrin from Life Technologies Corporation (Eugene, OR), and Histodenz from Sigma-Aldrich (St. Louis, MO). Dialysis tubing was from Repligen Corporation (Waltham, MA) and Bio-Beads SM-2 Resin from Bio-Rad Laboratories (Hercules, CA). Underivatized streptavidin and Pierce Protein-A Magnetic Beads were from Thermo Scientific (Waltham, MA).

Orr A., Song H, & Wickner W. (2022). Fusion with wild-type SNARE domains is controlled by juxtamembrane domains, transmembrane anchors, and Sec17. Molecular Biology of the Cell, 33(5), ar38.

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Lipid Preparation and Storage Protocol

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Reconstitution of Membrane Proteins

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PI3P was from Echelon (Salt Lake City, Utah), ergosterol from Sigma (St. Louis, MO), fluorescent lipids from Thermo Fisher (Waltham, MA), and other lipids were from Avanti (Alabaster, AL). Biobeads SM2 were from BioRad, Cy5-Streptavidin from SeraCare (Milford, MA), biotinylated phycoerythrin from Invitrogen (Eugene, OR) and underivatized streptavidin from Thermo Fisher. Spectrapor 6 dialysis tubing (7.5mm dia, 25 kDa cutoff) was from Spectrum Labs (Las Vegas, NV). Octyl--D-glucopyranoside was purchased from Anatrace (Maumee, OH).

Song H., Torng T., Orr A., Brünger A.T., & Wickner W. (2021). Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering.

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