PerkinElmer Phos-tools for phosphorylation analysis

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1 rotein Biology Reagents: hos-tools erkinelmer hos-tools for phosphorylation analysis erkinelmer hos-tools is a suite of reagent kits for sensitive and selective analysis of phosphorylation-based cell signaling. hos-tools kits incorporate novel chemistries with universal affinity for phosphorylated residues, replacing or supplementing antibodybased protocols. The phosphorylation of proteins is critical to regulating cell signaling and ultimately, the function of biological systems. Most cellular processes are regulated by reversible phosphorylation of proteins and upwards of 30% of the total complement of proteins expressed by human cells are likely to be phosphorylated at some point. Determination of a protein s phosphorylation state is important for identifying protein kinase substrates and revealing the on/off state of signal transduction pathways. hos-trap combines the selectivity of titania for phosphopeptides and the convenience of magnetic beads for superior sensitivity, selectivity and ease-of-use for subsequent MS analysis. hos-tag gel and blot stains incorporate a novel chelator with unprecedented phospho-selectivity. The chelator may be coupled to a variety of fluorophores to provide the best sensitivity with a wide range of imagers. Cell Lysate Selective Enrichment of hosphoproteins hos-tag Selective Staining In-Gel Digestion hos-trap hosphopeptide Enrichment MS Analysis hosphoprotein Enrichment & Detection 13 9 EGF-Stimulated A431 Cell Lysate rotein ID and Mapping of hosphorylation Sites by MS 2

2 hos-trap: efficient phosphopeptide enrichment for mass spectrometry hos-trap uses thin-film titania coated magnetic beads to effectively replace existing IMAC technologies for phosphopeptide enrichment. rocess 96 samples simultaneously in a microplate or one at a time in a microfuge tube Kits designed for compatibility with MALDI and ESI mass spectrometry Simple, rapid protocol designed to enable automation of workflow hos-trap benefits Enriches phosphopeptides directly from complex biological samples: protein digests, serum/ plasma/urine Enhanced sensitivity Enrich and detect phosphopeptides from < 100 fmol of phosphoprotein Much higher sensitivity than most conventional IMAC systems Ease of use Simple protocol performed in < 10 minutes Flexible and automatable kit format for 1-96 enrichments Stable coating does not leach metals into your sample hos-trap kit 2

3 Titania-coated Magnetic Bead hosphoprotein hosphoprotein digested into peptides hosphorylated peptides bind to titania-coated magnetic bead while non-phosphorylated peptides do not Low fmol sensitivity from digests High selectivity in complex matrices 78 fmol to 1250 fmol of -casein digest was fractionated using hos-trap hosphopeptide Enrichment Kit. 10 µl of normal human serum was fractionated using ros-trap hosphopeptide Enrichment Kit.

4 hos-tag gel and blot stains for detection of protein phosphorylation hos-tag is a novel chelate with unparalleled selectivity and sensitivity for phosphoproteins. It exhibits excellent affinity for the fluorescent detection of phosphomonoesters of tyrosine, serine and threonine. hos-tag can be linked to a variety of fluorophores, allowing a range of choices in spectral properties and detection hardware. erkinelmer offers a family of gel and blot stain kits based upon hos-tag. hos-tag benefits Antibody-like selectivity for phosphoproteins Binding affinity for phosphomonoesters in the nanomolar range Minimal affinity for acidic residues Sensitivity to ~1 ng phosphoprotein for gels, ~5 ng for blots comparable to total protein stains Linear dynamic range over 3 orders of magnitude Choice of excitation wavelengths for high sensitivity with any gel doc system hos-tag 300/460 for use with UV transilluminator (300 nm), blue LED laser (457 nm), SHG laser (473 nm), Argon laser (488 nm), Xenon lamp with 460 nm filter hos-tag 540 for use with Solid State YAG Laser (532 nm), HeNe Laser (543 nm), Xenon lamp with 540 nm filter Counterstain with total protein stain to assess level of phosphorylation protein abundance. No need to destain between steps Compatible with MALDI-TOF mass spectrometry The hos-tag structure incorporates a dinuclear zinc chelation site, mimicking natural phosphate binding domains. (Chem Rev Feb; 104(2): , J Biol Inorg Chem Apr; 5(2): , etc.) 4

5 1. hos-tag 540 phosphoprotein gel stain 2. Total protein stain Gel was loaded with 100 µg of protein from Rat Liver Cytosol. The IEF was carried out with ph ampholines for 9600 volt-hours. The tube gel was sealed to a 10% gel for the second dimension SDS-AGE. rotein 1 not detected by lysine and arginine specific total protein stain. hosphoproteins identified in gel images above. Accession # rotein Name 1 gi: Beta-1 syntrophin (known py) 2 gi: T-box 4 3 gi: MAKK 7 (known py) 4 gi: HOGG 1 (known ps) 5 gi: Decay accelerating factor soluble form percursor, CD55 hos-tag kit lease visit for technical information including posters and selected references. 5

6 Ordering information roduct Description roduct No. hos-trap hosphopeptide Enrichment Kit 96 Sample Enrichments RT301001KT hos-trap Kit contents: hos-trap Magnetic Beads, binding buffer, washing buffer, elution buffer, 6-well V-Bottom polypropylene microplate 9 and cover (2), manual. roduct Description roduct No. hos-tag 300/460 hosphoprotein Gel Stain For 20 mini-gels or two 2D gels RD400A001KT hos-tag 300/460 hosphoprotein Blot Stain For 16 mini-gel blots RD410A001KT hos-tag 540 hosphoprotein Gel Stain For 20 mini-gels or two 2D gels RD500A001KT hos-tag 540 hosphoprotein Blot Stain For 16 mini-gel blots RD510A001KT All hos-tag kits include stain concentrate, blocking buffer and stain buffer. Related roduct Description roduct No. 96-Well Magnet Toroid design deep well plate magnet for use with magnetic bead applications Cellular rotein Fractionation Kit For initial sample complexity reduction RD101A001KT erkinelmer, Inc. 940 Winter Street Waltham, MA USA hone: (800) or (+1) For a complete listing of our global offices, visit erkinelmer, Inc. All rights reserved. The erkinelmer logo and design are registered trademarks of erkinelmer, Inc. ExactTag, hos-trap and hos-tools are trademarks and erkinelmer is a registered trademark of erkinelmer, Inc. or its subsidiaries, in the United States and other countries. hos-tag is a trademark of the NARD Institute, Ltd. All other trademarks not owned by erkinelmer, Inc. or its subsidiaries that are depicted herein are the property of their respective owners. erkinelmer reserves the right to change this document at any time without notice and disclaims liability for editorial, pictorial or typographical errors A_03 rinted in USA