Akt Pathway Phospho 7-Plex Panel
Contents
1. KHO0461 The p70S6K pTpS421 424 was calibrated to Invitrogen Luminex kit catalog number LHO0191 One unit of IRS 1 pS312 is equivalent to 150 pg recombinant IRS 1 that was phosphorylated by JNK2al1 Invitrogen Cat PHO3035 One unit of IGF IR pYpY1135 1136 is equivalent to 17 pg autophosphorylated recombinant IGF 1R Subsequent lots of standard will be normalized to this lot of material to allow consistency of IRS 1 pS312 and IGF 1R pYpY1135 1136 quantitation Performance Characteristics Typical Standard Curves The standard curves presented here were collected as a 7 Plex but are depicted in two groupings for clarity Akt Pathway Phospho Seven Plex Standard Curves Akt Pathway Phospho Seven Plex Standard Curves o IR pYpY 1162 1163 IRS 1 12 IRS 1 pS312 m IGF 1R pYpY 1135 1136 a Akt pS473 7 t PRAS40 pT246 p70S6K pTp 421 424 x GSK 3b pS9 T T 150 200 250 300 100 150 200 250 300 350 400 450 units mL units mL Protein IGF 1R IRS 1 RAS40 p70S6K GSK 3f prvnemen B BE esas rssi DE e Sensitivity Sensitivity units mL lt 3 OO 24 4 4 lt 0 25 25 Recovery 10 8 103 2 100 9 5 9 p pepe a fm Recovery 10 102 104 9 109 8 103 101 pave eee Intra assay variation 5 6 7 3 5 7 6 0 6 3 6 1 4 5 woe Oe TO eee ee n 32 Analytical Sensitivity The analytical sensitivity was dete2. invit rogen Akt Pathway Phospho 7 Plex Panel Technical Data Sheet Catalog LHO0001 Lot 737526 Note A letter at the end of the lot number signifies an additional packaging of this same lot Intended Use This reagent set comprises of components for the measurement of the phosphorylated proteins IR pYpY1162 1163 IGF IR pYpY1135 1136 IRS 1 pS312 Akt pS473 PRAS40 pT246 p70S6K pTpS421 424 and GSK 3B pS9 in cell lysates and tissue homogenates These reagents are intended for use in the Luminex 100 or 200 System This kit is configured for research use only and is not to be used in diagnostic procedures Reagents Provided Akt Pathway Phospho 7 Plex l Part LM114 Description Antibody Bead Concentrate 10X Lot 737535 Size 0 25 mL 100 tests Analyte Bead Region Analyte Bead Region IR pYpY1162 1163 I2 PRAS40 pT246 74 IGF IR pYpY1135 1136 73 p70S6K pTpS421 424 58 IRS 1 pS312 75 GSK 3 pS9 59 Akt pS473 14 Form 0 25 mL 10X bead concentrate solution in storage buffer Contains 0 05 sodium azide as a preservative Storage Light sensitive material Store at 2 to 8 C in the dark until the expiration date indicated on the kit Recommended Dilution Dilute 2 5 uL Antibody Bead Concentrate in 25 uL Working Wash Solution per assay well Use 25 uL of diluted bead solution per well Refer to the User Manual included in the kit for further information Akt Pathway Phospho 7 Plex 2 Part DNI
3. products are for research use only and are not intended for human or animal diagnostic therapeutic or commercial use
4. 430 units mL GSK 36 pS9 160 units mL Akt pS473 140 units mL Tmportant note The concentrations of reconstituted standards are lot specific Please verify all concentration values entered in data analysis software Reconstitution Reconstitute in 0 5 mL Assay Diluent For Research Use Only CAUTION Not for human or animal therapeutic or diagnostic use Invitrogen Corporation 542 Flynn Rd Camarillo e CA 93012 Tel 800 955 6288 E mail techsupport invitrogen com PILHO0001 Rev 03 10 Page 1 of 3 DCC 10 0814 Important Licensing Information These products may be covered by one or more Limited Use Label Licenses see the Invitrogen Catalog or our website www invitrogen com By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses Unless otherwise indicated these products are for research use only and are not intended for human or animal diagnostic therapeutic or commercial use Akt Pathway Phospho 7 Plex Panel Technical Data Sheet Recommended Starting Concentration for Standard Curve Upon reconstitution the starting concentration of standards is the value cited above Make 2 fold serial dilutions in Assay Diluent Refer to the User Manual included in the kit for further information Calibration The following markers were calibrated to Invitrogen ELISA kits catalog numbers IR pYpY1162 1163 KHR9131 Akt pS473 KHOO111 PRAS40 pT246 KHO0421 and GSK 3B pS9
5. 14 Description Detection Ab Concentrate 10X Lot 737534 Size 0 5 mL 100 tests Form 0 500 mL of a 10X stock of Detector Antibody Concentrate Contains 0 1 sodium azide as preservative Concentration of antibody is matched to this lot of beads Do not mix lots of Coated Beads and Detector Antibody Storage Store at 2 to 8 C until the expiration date indicated on the kit Recommended Dilution Dilute 5 uL Detector Antibody Concentrate in 50 uL Detector Antibody Diluent per assay well Use 50 uL of diluted detector antibody per well Refer to the User Manual included in the kit for further information 3 Part SM040 Description Akt Pathway Phospho 7 Plex Standard Lot 774545 Size 2 Vials Form This standard lyophilized recombinant proteins is designated in units mL The proteins in this standard have been calibrated with the respective Invitrogen ELISA kits when available Detailed information on calibration is provided on the accompanying page Contains 0 1 sodium azide as a preservative Storage Store at 2 to 8 C Use within 1 hour after reconstitution The remaining reconstituted standard should be discarded or frozen in aliquots at 80 C for further use The standard can be frozen and thawed one time without loss of immunoreactivity Concentration of Reconstituted Standards IR pYpY 1162 1163 830 units mL PRAS4O0 pT246 700 units mL IGF 1R pYpY1135 1136 660 units mL p70S6K pTpS421 424 320 units mL IRS 1 pS312
6. HO T cells Figure B were treated with insulin Cells were serum starved for 15 hours and either left untreated or treated with 100 nM of insulin for 15 minutes In figure C HepG2 cells were stimulated by IGF1 in the presence of sodium vanadate In this study HepG2 cells were serum starved for 15 hours and either left untreated or treated with 100 ng mL of IGF1 for 15 minutes In figure D Jurkat cells were treated with Wortmannin a PI3 kinase inhibitor The cells were either left untreated or treated with 200 nM of Wortmannin for 3 hours A C 10000 4000 8000 m Non treated m Non treated m Insulin 3000 m Vanadate IGF1 6000 2000 4000 2000 I 1000 0 m A l _ i 0 a I l m IR IGF 1R IRS 1 Akt par p70S6K GSK 3B IR IGF 1R IRS 1 Akt PRAS40 p 0S6K GSK 3B B D 6000 8000 m Non treated m Non treated 4500 ainsin 6000 m Wortmannin L 3000 4000 1500 2000 0 ie I 0 nm oa a IGF 1R IRS 1 PRAS40 p 70S6K GSK 3B IR IGF 1R IRS 1 Akt PRAS40 p 70S6K GSK 3B Sample Preparation This kit has been validated with cell lysates prepared in Cell Extraction Buffer Invitrogen Cat FNN0011 and NP40 Cell Lysis Buffer Invitrogen Cat FNN0021 Optimization of cell stimulation and cell lysis procedures may be required for each specific application Refer User Manual for detailed instructions Explana
7. rmined by adding two standard deviations to the median fluorescence intensity obtained when the zero standard was assayed 32 times Recovery To evaluate recovery analytes were spiked at 3 different concentrations into 10 Cell Extraction Buffer Invitrogen Cat FNNOOI1 and 10 NP40 Cell Lysis Buffer Invitrogen Cat FNN0021 Species Reactivity Each analyte specific component of this reagent set recognizes human mouse and rat proteins Other species have not been tested For Research Use Only CAUTION Not for human or animal therapeutic or diagnostic use Invitrogen Corporation 542 Flynn Rd Camarillo e CA 93012 Tel 800 955 6288 E mail techsupport invitrogen com PILHO0001 Rev 03 10 Page 2 of 3 DCC 10 0814 Important Licensing Information These products may be covered by one or more Limited Use Label Licenses see the Invitrogen Catalog or our website www invitrogen com By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses Unless otherwise indicated these products are for research use only and are not intended for human or animal diagnostic therapeutic or commercial use Akt Pathway Phospho 7 Plex Panel Technical Data Sheet Specificity The analytes of the multiplex were assayed individually with the Akt Pathway Phospho 7 Plex and were all found to have no cross reactivity To further evaluate the performance of this kit MCF7 cells Figure A and IR transfected C
8. tion of symbols Le Catalogue Number Batch code REO Research Use Only In vitro diagnostic medical device Manufacturer EC REP European Community authorised representative Ho Without does not contain With contains S pine Protect from light AN Consult accompanying documents k i prees the user to consult instructions for use IFU accompanying the product By purchasing this Kit which contains fluorescently labeled microsphere beads authorized by Luminex Corporation Luminex you the customer acquire the right under Luminex s patent rights to use this Kit or any portion of this Kit including without limitation the microsphere beads contained herein only with Luminex s laser based fluorescent analytical test instrumentation marketed under the name Luminex 100 or 200 This product is covered by one or more of the following U S patents 6 046 807 For Research Use Only CAUTION Not for human or animal therapeutic or diagnostic use Invitrogen Corporation 542 Flynn Rd Camarillo e CA 93012 Tel 800 955 6288 E mail techsupport invitrogen com PILHOO0001 Rev 03 10 Page 3 of 3 DCC 10 0814 Important Licensing Information These products may be covered by one or more Limited Use Label Licenses see the Invitrogen Catalog or our website www invitrogen com By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses Unless otherwise indicated these
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