User Manual
Contents
1. 10 9 8 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 19 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Limitations of the Procedure Do not calculate the concentrations of analytes if the MFI value is higher than the highest standard or lower than the lowest standard If measured analyte concentrations exceed the value of the upper range of the standard curve dilute samples appropriately and reanalyze Dilute serum and plasma samples in Assay Diluent Dilute tissue culture supernatants in tissue culture medium To measure samples with very low analyte levels it may be possible to extend the range at the low end of the curve by additional dilution of Standard 7 The influence of drugs or other substances not usually found in serum or plasma have not been investigated The influence of abnormal jaundiced hemolyzed lipemic sera or plasma samples has not been investigated The rate of degradation of analytes in serum plasma or tissue culture supernatants has not been investigated The affect of heterophilic antibodies on the assay has not been investigated 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 20 d ORIGENE True2. e The XY platform heater should be off e The total number of beads to count is 1100 e The minimum events setting should be 30 e The sample size should be set to 50 pL e The flow rate should be set to Fast 4 Enter the analyte names and bead numbers as indicated in the Table 1 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 15 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company 5 Check the probe height and adjust it if necessary to accommodate the filter plate 6 Perform 1 prime with sheath fluid 1 alcohol flush and 2 sheath fluid washes 7 Calibrate to High Gain setting if necessary see page 10 Preparation of Detection Reagent Streptavidin Phycoerythrin Conjugate e Prepare Detection Reagent according to the table below Volume per Number of Final Component Reaction Wells Volume Assay Buffer B 90 uL SAPE Conjugate 10 uL Detection with Streptavidin Phycoerythrin 1 Remove the liquid in the wells by gentle vacuum 2 Wash beads three times by adding 100 ul Wash Buffer Il to all wells and removing liquid with gentle vacuum 3 Blot plate thoroughly on absorbent material 4 Add 100 yl Detection Reagent Streptavidin PE conjugate into each well 5 Cover and incubate the plate for 30 minutes at room temperature on an orbital plate shaker at 600 800 rpm 6 Wash beads thre
3. 15 Sealers Creating Custom Multiplexes Custom multiplex assays can be prepared by purchasing one or more single plex reagent sets and the Core Reagent Kit Each of the single plex reagent sets contains beads detection antibody and protein standard Up to ten beads and detection antibodies can be mixed to create a custom multiplex see Appendix I The protein standard is pre mixed The Core Reagent Kit contains all of the buffers and accessories necessary to run the assay with a single plex reagent set or a custom multiplex 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 3 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Important Information e Store the reagents at the temperature and condition specified on the labels e Read the entire protocol before use e Research Use Only The product you have received is authorized for laboratory research use only The product has not been qualified or found safe and effective for any human or animal diagnostic or therapeutic application Uses other than the labeled intended use may be a violation of applicable law e Hazards It is the end user s responsibility to consult the applicable MSDS s before using this product Disposal of waste materials must comply with all appropriate federal state and local regulations If you have any questions concerning the h
4. Absorbent paper towels for example Wypall X60 from Kimberly Clark 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 5 ORIGENE Your Gene Company Equipment and software required Description Luminex 100 200 or equivalent Luminex Data Acquisition Software Vortex Mixer Mini centrifuge Orbital Shaker Water Bath Sonicator Microplate vacuum manifold with pressure gauge Data Analysis Software 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com TruePLEX Human Growth Factor Panel 11 Plex Recommended Supplier Cat No Luminex Millipore Hitachi Bio Rad XPONENT 3 1 Luminex IS 2 3 Luminex LDS1 7 BioPlex Manager VWR Analog Vortex Mixer 58816 121 120V 58816 123 230V VWR Minifuge 93000 196 120V 93000 198 230V Eppendorf Mix Mate 022674200 120V 60Hz 022674226 230V 50Hz Bransonic Model B3 B5 000 951 005 Model B3 115V 000 951 103 Model B5 230V Pall Multi well plate vacuum manifold PN 5017 MasterPlex QT from Hitachi Software or equivalent Page 6 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Overview and Intended Use OriGene s TruePlex immunoassay kits are designed to measure the concentration of one or more proteins in a complex biological
5. Final Volume Size Assay Buffer each Det Ab of Det Ab ul B to add pl to Add ul Added ul 1 90 10 10 100 2 80 10 20 100 3 70 10 30 100 4 60 10 40 100 5 50 10 50 100 6 40 10 60 100 7 30 10 70 100 8 20 10 80 100 9 10 10 90 100 10 0 10 100 100 11 Must purchase 11 plex kit 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 24
6. a plate plan be designed Such a plan will assist in assay workflow and data analysis A suggested plate plan is shown below A plate plan template is provided on page 19 1 2 3 5 6 7 8 9 10 11 12 IO nm cg oIm gt Std 1 is the highest concentration and Std 7 is the lowest concentration Running all standards samples and controls in duplicate or triplicate is recommended 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 11 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Sample Collection and Preparation 1 Serum plasma and tissue culture supernatants are suitable for use in this assay Additional sample types may also be suitable but have not been validated with this assay 2 Avoid the use of lipemic of hemolyzed samples 3 Collect samples according to standard protocols immediately mix and separate and then aliquot the samples into polypropylene tubes If not tested immediately store samples at 80 C 4 Analyze fresh samples as quickly as possible after collection Allow frozen samples to thaw on ice then mix well Avoid multiple freeze thaw cycles of frozen samples 5 Turbid samples should be clarified by centrifugation before use 6 If measured analyte concentrations exceed the value of the upper range of the standard curve dilute sam
7. reader The sensitivity linearity and dymanic range of OriGene s TruePlex immunoassays are comparable to traditional ELISA assays performed in a microplate OriGene s TruePlex Human Growth Factor 11 Plex Kit is designed for the measurement of eleven different growth factors in cell culture supernatant human sera or plasma Visit the OriGene website at www origene com immunoassay luminex for a current listing of available TruePlex assays and reagents and for information regarding custom assay development Assay Principles OriGene TruePlex immunoassays use Luminex xMAP technology to enable the efficient multiplexing of up to 100 different assays for simultaneous analysis Luminex xMAP technology utilizes color coded 5 6 um beads that have been internally dyed with two different fluors By varying the concentration of fluors within each bead the beads can be easily distinguished by the Luminex analyzer 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 7 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company For immunoassays capture antibodies for each target analyte are on 00009239 bound to a specific bead type or color Different bead types can 0022225000 then be mixed to create defined multiplexes Beads and samples 22200209222 are added to a 96 well filter plate and allowed to incubate During 0022222500 t
8. 00 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 2 ORIGENE Your Gene Company Ordering Information TruePLEX Human Growth Factor Panel 11 Plex Catalog Description Quantity Number AM100096 TruePLEX Human Growth Factor Assay Kit 11 Plex 96 Reactions AM100097 TruePLEX Extracellular Core Reagent Kit 96 Reactions AM100098 TruePLEX PDGF AB Single Plex Reagent Set 96 Reactions AM100099 TruePLEX PDGF BB Single Plex Reagent Set 96 Reactions AM100100 TruePLEX FGF 4 Single Plex Reagent Set 96 Reactions AM100101 TruePLEX VEGFD Single Plex Reagent Set 96 Reactions AM100102 TruePLEX FGF 2 Single Plex Reagent Set 96 Reactions AM100103 TruePLEX EGF Single Plex Reagent Set 96 Reactions AM100104 TruePLEX HGF Single Plex Reagent Set 96 Reactions AM100105 TruePLEX FLT3LG Single Plex Reagent Set 96 Reactions AM100106 TruePLEX ANGPT2 Single Plex Reagent Set 96 Reactions AM100107 TruePLEX PGF Single plex Reagent Set 96 Reactions AM100108 TruePLEX VEGFA Single plex Reagent Set 96 Reactions AM100109 TruePLEX Detection Reagent 2 x 96 Rxns AM100110 TruePLEX Wash Buffer Set 2x 10 ml Wash Buffers and II 2x25ml AM100111 TruePLEX Assay Buffer Set 2 x 10 ml Assay Diluent Assay Buffer A Assay Buffer B 2 x 10 ml 2 x 50 ml AM100112 Filter Plates with Sealers Pk 5 plates
9. PLEX Human Growth Factor Panel 11 Plex Your Gene Company Troubleshooting To troubleshoot problems with the Luminex instrument consult the appropriate Luminex manuals contact Luminex technical support http www luminexcorp com support or contact your instrument supplier To troubleshoot problems with the using MasterPlex QT for data analysis contact Hitachi Software http www miraibio com support Problem Cause Insufficient Bead mix not prepared correctly bead count Vacuum pressure too high Plate leaked in the Luminex instrument Clogged sample probe Wells in filter Clogged wells plate will not vacuum Plate has not made a tight seal with the vacuum manifold Leaking plate Probe Height not adjusted correctly Insufficient blotting of filter plate Vacuum pressure too high High Cross contamination of wells Background Ineffective or omitted wash steps 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Solution Prepare new bead mix and re run samples Sonicate and vortex vial containing 10X Bead Mix Adjust vacuum pressure to 2 5 mm Hg during filtration See below Clean sample probe Remove and sonicate if necessary Spin samples 14 000 x g for 2 minutes before removing aliquot for testing Replace gasket on vacuum manifold Adjust probe height with two alignment disks using the filter p
10. azards associated with this product please call OriGene Technologies Inc at 1 888 267 4436 e Terms and Conditions By opening the packaging containing this Assay Product which contains fluorescently labeled microsphere beads authorized by Luminex Corporation or using this Assay Product in any manner you are consenting and agreeing to be bound by the following terms and conditions You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you If you do not agree to all of the terms and conditions set forth below you must return the product unopened within ten 10 day of receipt under the same shipping conditions as received to receive a full refund You the customer acquire the right under Luminex Corporation s patent rights if any to use this Assay Product or any portion of this Assay Product including without limitation the microsphere beads contained herein only with Luminex Corporation s laser based fluorescent analytical test instrumentation marketed under the name Luminex 100 200 e Safety and Use All biological materials should be handled as potentially hazardous Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of potentially infectious or hazardous agents This product is authorized for laboratory research use only The
11. ch well Remove liquid from wells by gentle vacuum Blot thoroughly Add 100 ul 1X Bead Mix to each well Remove liquid from wells by gentle vacuum Blot thoroughly Add 100 ul of Capture Buffer to all wells Add 50 ul of standards or samples into designated wells Cover and incubate for 2 hours with shaking 600 800 rpm Prepare 1X Detection Antibody EN saa o a gt a N Remove liquid from plate by gentle vacuum Wash two times with 100 ul 1X Wash Buffer 10 Wash one times with 100 ul 1X Wash Buffer Il Blot thoroughly 11 Add 100 pl of 1X Detection Antibody to each well 12 Cover plate and incubate for 1 hour with shaking 600 800 rpm 13 Prepare Luminex instrument for reading 14 Prepare Detection Reagent 15 Remove liquid from wells by gentle vacuum 16 Wash three times with 1X Wash Buffer Il Blot thoroughly 17 Add 100 yl 1X Detection Reagent to each well 18 Cover and incubate for 30 minutes with shaking 19 Wash three times with 1X Wash Buffer II 20 Remove liquid from wells by gentle vacuum Blot thoroughly 21 Add 100 ul of 1X Wash Buffer Il to each well 22 Shake for 2 minutes 600 800 rpm 23 Read in Luminex instrument 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 18 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Plate Setup Template 12 11
12. d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company APPLICATION GUIDE TABLE OF CONTENTS DrOering TATE TIAN 2 m line 3 Creating Custom Multiplexes m 3 Components supplied with this kit un en 5 seriei m en 5 Materials required but not provided eeeeeeeeee rnnnnnnnnnnnnnnnnnnnnnnnnannnnnnnnnnnnnnnn 5 Equipment and software required Laman oras xat exi on d XE s SERE QI und 6 Assay PEnCIDIGS iain aie see sid iG cn annie cnn caesar sien ena IU KE RUD UU INE AERE 7 PTOBEALIFE OUT e e E 9 Setting the Luminex 100 or 200 for High Gain of High PMT Reading 10 Procedure NOUS e ae 10 Bead ASSIgNMEN S GAGANA 11 Recommended Plate Layout u lb bb a C aD Tan da a AGA 11 Sample Collection and Preparation uiris ornetur tace een 12 REAGENT Preparation uma tinis ink prac UO GU EA ED GARA 12 1X Wash Buffer Hi ete ana ER RR E ER a d 12 1X Wash Bunter e ANNA NAA rn 12 Standards for serum or plasma samples 0000707777000000000000000000EEEEEEERAEEEAEEERAEER ARKA AKA rnnt 13 Standards for cell culture supernatants 0 00000000000000000000000000E0EEEEANEKASERASEE AE KE AREA RANA rentrer 13 Capture Buffer fo
13. dard 7 1 3 and re run the sample and standard curve Dilute samples in Assay Diluent and re run Page 22 TruePLEX Human Growth Factor Panel 11 Plex ORIGENE Your Gene Company Appendix I Creating multiplexes from single plex reagents Custom multiplexes can be prepared from single plex reagents by mixing the 10X Beads and 10X Detection Antibody from the single plex kits into a single 1X Mix Use the tables below to calculate the volumes of 10X Beads 10X Detection Antibody and Assay Buffer B to mix to create the respective 1X mixes Note that all volumes are given on a per well basis Multiply given volumes by the number of wells to be run Add one or two additional wells to ensure a sufficient volume of the 1X mixes 1X Bead Mix Volume of Volume of Total Volume Ka Assay Buffer each Bead to of Beads Final une B to add pl Add pl Added ul 1 90 10 10 100 2 80 10 20 100 3 70 10 30 100 4 60 10 40 100 5 50 10 50 100 6 40 10 60 100 7 30 10 70 100 8 20 10 80 100 9 10 10 90 100 10 0 10 100 100 11 Must purchase 11 plex kit 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 23 ORIGENE Your Gene Company 1X Detection Antibod TruePLEX Human Growth Factor Panel 11 Plex Plex Volume of Volume of Total Volume
14. e times by adding 100 ul Wash Buffer II to all wells and and then removing liquid with gentle vacuum 7 Blot plate thoroughly on absorbent material 8 Add 100 uL 1X Wash Buffer Il to each well 9 Shake plate for 2 minutes on an orbital plate shaker at 600 800 rpm 10 Read plate in the Luminex instrument 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 16 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Data Analysis using MasterPlex QT 1 A free two week trial of MasterPlex QT from Hitachi software is available for quantitative analysis of your data If you don t already have a copy download a copy of MasterPlex QT from www miraibio com Follow the instructions for installing the software and licenses 2 MasterPlex QT Tutorials in PDF format PowerPoint presentations FAQs and other training materials are also available on the MiraiBio website 3 The standard curves of the Growth Factor Panel generally give the best fit with a 5 parameter logistic 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 17 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Protocol Summary Prepare 1X Wash Buffer and Il Standards 1X Bead Mix and Capture Buffer Add 100 ul Wash Buffer Il to ea
15. ent wash steps only use Wash Buffer II 3 The vacuum pressure on the vacuum manifold should not exceed 5 mm Hg Optimal filtration occurs between 2 and 4 mm Hg 4 The fluorescent beads are light sensitive and are especially sensitive to direct sunlight and incandescent light Use aluminum foil to cover plates and tubes Store plates in a dark location during incubations 5 After vacuum filtration blot plates on clean absorbent material such as WYPALL X60 towels from Kimberly Clark 6 During loading or incubations do not place the filter plate on absorbent material as liquid will be drawn through the plate by wicking 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 10 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Bead Assignments Analyte Name Symbol ELE Eie om Platelet derived growth factor AB PDGFAB 68 5 Platelet derived growth factor BB PDGFBB 73 10 Fibroblast growth factor 4 FGF4 75 20 Vascular endothelial growth factor D VEGFD 78 20 Fibroblast growth factor 2 FGF2 79 10 Epidermal growth factor EGF 80 5 Hepatocyte growth factor HGF 86 10 FIt 3 ligand FLT3LG 89 5 Angiopoietin 2 ANGPT2 90 20 Placental growth factor PGF 91 5 Vascular endothelial growth factor A VEGFA 100 20 Recommended Plate Layout Before starting it is recommended that
16. f Bead Mix Concentrate TruePLEX Human Growth Factor Panel 11 Plex e Sonicate the Bead Mix Concentrate for 30 seconds e Prepare 1X Bead Mix according to the table below Component Volume per Number of Final P Reaction Wells Volume Assay Buffer B 90 uL Bead Mix 10 uL e Mix thoroughly by inversion Protect from light by covering tube in aluminum foil Filter Plate Before starting the assay cover the wells that will not be used in the assay with an aluminum plate sealer supplied Press sealer down so that all wells are tightly sealed Trim edges with a razor blade Assay Protocol Analyte Capture 1 Cover unused wells with an aluminum plate sealer 2 Add 100 yl Wash Buffer Il to each of the wells that will be used 3 Remove liquid from the wells by gentle vacuum do not exceed a vacuum pressure of 5 mm Hg Blot plate thoroughly on absorbent paper 4 Briefly vortex the 1X Bead Mix Add 100 pl of 1X Bead Mix to each assay well Remove liquid from the plate by gently vacuum Blot plate thoroughly on absorbent paper CON Oo 0 Add 50 ul sample into designated sample wells Add 100 yl Capture Buffer into all standard blank and sample wells Add 50 ul standards into designated standard wells including negative Cover and incubate the plate for 2 hours at room temperature on an orbital plate shaker 600 800 rpm 9 Ten to fifteen minutes before the end of this incubation prepare 1X Detect
17. he incubation target analytes are captured onto the bead surfaces 2220022222 by the bound antibodies Following a wash step a mixture of 99999 2 000002 biotinylated detector antibodies are added and allowed to incubate 2220000099 with the beads The biotinylated detector antibodies subsequently bind to captured target analytes After removal of excess biotinylated detector antibodies streptavidin phycoerythrin conjugate Detection Reagent is added to the beads After incubation and another wash the beads are read in the Luminex instrument The fluorescent intensity of the phycoerythrin bound to each bead is proportional to the amount of captured analyte Recombinant protein standards are used to generate standard curves for each analyte and protein levels in each sample are determined by interpolation of the signals from for each analyte 35000 PDGF B 30000 EGF Fit 3 25000 ese FGF2 20000 PDGF R 15000 HGF VEGF D 10000 Ang 2 FGF4 5000 VEGF A Median Fluorescent Intensity MFI 0 1 1 10 100 1000 10000 Analyte Concentration pg ml 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 8 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Procedure Outline Pre wet Filter Plate Add beads to Filter Plate Add Buffer Samples Stds Incubate 2 hours Add Detec
18. ion Antibody 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 14 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Preparation of 1X Detection Antibod e Prepare Detection Antibody according to the table below Combonant Volume per Number of Final P Reaction Wells Volume Assay Buffer B 90 uL Detection Antibody 10X 10 uL Analyte Detection 1 Remove the liquid from the wells by gentle vacuum 2 Wash beads two times by adding 100 uL Wash Buffer to the wells and removing liquid with gentle vacuum 3 Wash beads one times by adding 100 uL Wash Buffer Il to the wells and removing liquid with gentle vacuum After washing blot plate thoroughly on absorbent material 4 Add 100 yl Detection Antibody into each well 5 Cover and incubate the plate for 1 hour at room temperature on an orbital plate shaker at 600 800 rpm 6 During this incubation prepare the Luminex instrument for reading see below 7T During the last 10 minutes of this incubation prepare the Detection Reagent Streptavidin Phycoerythrin Prepare the Luminex instrument for reading 1 Set up the instrument as described in the user s manual Instruments from different vendors may have different set up procedures 2 Warm up the instrument This may take up to 30 minutes 3 Parameters specific to this kit
19. ix well by inversion a minimum of 6 times Use Standard 1 as supplied Dilute Standard 1 as follows to prepare the remaining standards To Make Add To Standard 2 150 ul of Standard 1 200 ul of Assay Diluent Standard 3 100 ul of Standard 2 200 ul of Assay Diluent Standard 4 100 ul of Standard 3 200 ul of Assay Diluent Standard 5 100 ul of Standard 4 200 ul of Assay Diluent Standard 6 100 ul of Standard 5 200 ul of Assay Diluent Standard 7 100 ul of Standard 6 200 ul of Assay Diluent Use Assay Diluent as the Negative Standard blank Standards for cell culture supernatants Prepare as above but use cell culture medium or other matrix in the place of Assay Diluent Use cell culture medium or other matrix as the Negative Standard blank Capture Buffer for serum or plasma samples Prepare the following buffer just before use Prepare enough for several extra wells to account for losses during pipetting Volume per Number of Final Component Reaction Wells Volume Assay Buffer A 65 uL Assay Diluent 35 uL Capture Buffer for cell culture supernatants Prepare as above but use cell culture medium or other matrix in the place of Assay Diluent 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 13 ORIGENE Your Gene Company 1X Bead Mix e Briefly vortex 5 seconds the vial o
20. late in the kit Blot filter plate thoroughly onto paper towels after each washing cycle Ensure that the vacuum pressure is less than 5 mm Hg Pipette carefully to ensure that no material from the standard or sample wells reaches the background Neg wells Follow wash instructions carefully Increase number of washes if necessary Page 21 ORIGENE Your Gene Company Problem Low signal across the plate Beads not in region Sample readings are out of range Cause Luminex instrument gain setting is incorrect 1X Detection Antibody prepared incorrectly Incubations were too short or shaking was insufficient Instrument is out of calibration Beads are photo bleached Incorrect bead regions entered Analyte level is below the detection limit of the assay Analyte level is above of the quantifiable range of the assay 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com TruePLEX Human Growth Factor Panel 11 Plex Solution Make sure that the reporter channel is calibrated to the High Gain or High PMT setting Prepare new 1X Detection Antibody Check assay conditions and remedy as necessary Recalibrate instrument Protect beads from light at all times Use amber colored tubes Check protocol template Correct if necessary Prepare an additional low standard by diluting Stan
21. ples appropriately and reanalyze Dilute serum and plasma samples in Assay Diluent Dilute tissue culture supernatants in tissue culture medium Reagent Preparation 1X Wash Buffer Prepare 1X Wash Buffer by diluting the entire contents of the 5X Wash Buffer bottle with 40 ml of sterile deionized water If desired add 20 mg sodium azide to bring the sodium azide concentration to 0 05 7 5 mM Store diluted Wash Buffer at 2 8 C Alternatively a portion of the 5X Wash Buffer can be diluted Mix 1 volume 5X Wash Buffer with 4 volumes of deionized water 1X Wash Buffer II Prepare 1X Wash Buffer Il by diluting the entire contents of the 20X Wash Buffer II bottle with 475 ml of sterile deionized water If desired add 0 24 g sodium azide to bring the sodium azide concentration to 0 0596 7 5 mM Store diluted Wash Buffer II at 2 8 C Alternatively a portion of the 20X Wash Buffer Il can be diluted Mix 1 volume 20X Wash Buffer II with 19 volumes of deionized water 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 12 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Standards for serum or plasma samples Dilute one vial of Standard just prior to use Do not store diluted standards Additional standards may be purchased from OriGene Thaw the Standard vial at room temperature for 20 minutes M
22. product has not been qualified or found safe and effective for any human or animal diagnostic application Uses other than the labeled intended use may be a violation of applicable law 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 4 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Components supplied with this kit Component Vol for 96 Rxns Human Growth Factor Antibody Bead Mix Concentrate 10X 960 uL Biotinylated Detection Antibody Concentrate 10X 960 uL Human Growth Factor Standard 11 Plex 3 x 500 uL Assay Diluent 10 mL Assay Buffer A 10 mL Assay Buffer B 50 mL Wash Buffer 5X 10 mL Wash Buffer II 20X 25mL Detection Reagent Streptavidin phycoerythrin 10X 960 uL Filter Plate 1 Plate Sealers 3 Storage Instructions The kit is shipped on blue ice The kit provides three vials of the Growth Factor Standards Upon receipt store the standards at 20 C or 80 Each vial is used once The following components may be stored at room temperature o Filter Plate o Plate Sealers All other components should be stored at 2 8 C Materials required but not provided Calibrated adjustable micropipettors with disposable plastic tips An 8 well multichannel pipettor is recommended but not required De ionized or molecular biology grade water Propylene tubes
23. r serum or plasma samples 107777700772200000000 ama nananana nananana nennen nanana nananana 13 Capture Buffer for cell culture supernatants 277700000000 aaa anan Nanana nananana rnnt 13 a AA AA APOY 14 giauur i MTMTTUMMM 14 Assay Protocol Pump a ee 14 Analyte Capture aaa ANG 14 Preparation or 1X Detection Antibody uus ete a na aka 15 Analyte DG LEC eL er 15 Prepare the Luminex instrument for reading urrs444444444444H0Hnnnnnnnnnnnnnnnnnnnnnnnnnnnannnnnnnnnn nhan nitent 15 Preparation of Detection Reagent Streptavidin Phycoerythrin Conjugate sesssssssss 16 Detection with Streptavidin Phycoerythrin u s aan 16 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 1 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Data Analysis using MasterPlex QT 5 6 cubitos o en tothp ee ee n Rp eth tte NAKAKA Baba Miete deren 17 bd koi elo Ez ID RD ERE TOC D LO AA 18 Plate Setup Template qoc M 19 Bine isinan 21 Appendix I Creating multiplexes from single plex reagents serere 23 1X Bead p e rr EUIS 23 1X Detection AO DY en P ately 24 9620 Medical Center Drive Suite 2
24. sample The kit reagents are intended to be used in conjunction with the Luminex 100 or 200 multianalyte flow analyzers manufactured by Luminex Corporation and sold by Invitrogen Bio Rad Millipore and others Advances in genomics proteomics and cell biology have allowed researchers to discover and characterize a wide range of complex extracellular and intracellular biological pathways Researchers studying these pathways have also uncovered multiple deviations from these pathways are often found in diseases and particularly cancer These deviations are often manifested by profound increases or decreases in levels of gene expression and concomitant changes in protein levels Immunoassays are the traditional tools for measuring protein levels in biological systems However traditional microplate assays can be expensive and slow Multiplexed bead based systems based on Luminex technology can dramatically increase the efficiency of protein measurements at reasonable cost OriGene s TruePlex immunoassays replace the solid phase microplates used in traditional immunoassays with color coded latex beads Each bead color or type can be coated with a specific antibody or antigen to make a specific assay Assays can be run as single plexes or may be combined in multiplexes according to a user s needs The assay is performed in a 96 well plate and the results are generated with the Luminex 100 or 200 instruments instead of a a traditional microplate
25. tion Antibody Incubate 1 hour Add Detection Reagent Incubate 30 minutes Add Wash Buffer Resuspend Beads Read in Luminex 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax techsupport origene com www origene com Page 9 d ORIGENE TruePLEX Human Growth Factor Panel 11 Plex Your Gene Company Note To achieve optimal results this kit requires the use of the High Gain of High PMT setting on the Luminex 100 or 200 Some instruments may use this setting as the default Check with your instrument supplier for more information Setting the Luminex 100 or 200 for High Gain of High PMT Reading 1 Create a new lot number for Cal 2 and enter it into the Luminex software as a new Cal 2 lot number use the actual lot number with an HG at the end to designate High Gain 2 Record the Cal 2 target value which is usually around 3800 3 Multiply the Cal 2 target value by 4 55 to get a new Target value of approximately 17 290 4 Enter the new Target Value as the value for your New Cal 2 lot 5 Run the Cal 2 Calibration Procedure Notes 1 Before mixing beads to create a custom multiplex ensure that each assay analyte is formatted onto a separate bead region 2 Two different wash buffers and Il are used for the first wash step after analyte capture The use of these two wash buffers minimizes potential clogging of the filter plate Subsequ
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