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1. Genemed Biotechnologies Inc Profiler Array Nonradioactive System User Manual Version 2 2 STORAGE Store Box 1 at room temperature RT Box 2 at 20 C and Box 3 at 4 C Please note that the Substrate is light sensitive and should be stored in the dark FOR RESEARCH USE ONLY PRT0088 Profiler Array Nonradioactive System TABLE OF CONTENTS L Introduction 3 IL Package Contents 5 IIl Additional Materials amp Equipment 6 IV Instructions for Use 6 V Troubleshooting Guide 10 Appendix 11 TRADEMARKS Profiler is a trademark of Genemed Biotechnologies Inc CDP ar is a registered trademark of Tropix Inc GenBank is a registered trademark of The United States Department of Health and Human Services FluorChem is a trademark of Alpha Innotech Corporation Hyperfilm is a trademark of Amersham Pharmacia Biotech Saran Wrap is a registered trademark of S C Johnson WARRANTY GENEMED BIOTECHNOLOGIES INC WARRANTS THAT THE MATERIALS SOLD MEET GENEMED S PERFORMANCE SPECIFICATIONS FROM THE TIME OF SHIPMENT UNTIL THE EXPIRATION DATE IF STORED UNDER THE RECOMMENDED CONDITIONS NO OTHER WARRANTIES OR GUARANTEES EXPRESSED OR IMPLIED ARE PROVIDED IN CLUDING WARRANTIES FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PUR POSE UNDER NO CIRCUMSTANCES SHALL GENEMED BE LIABLE FOR ANY DAMAGES ARISING OUT OF THE USE OF THE MATERIALS 2000 Genemed Biotechnologies Inc Page2 Genemed Bi2. weg ouv iwiso 385 1 ie 91 11 4494 esdgg fta umoo SAMIN savna LIV puer dse5 1 Nwaivod ggi 159 11 12 711 0494 s1v 3N 199501 aAu g zang eoXd LYE Sdov dseo jedy 9 osy fosas oza 9 1 494 19 r1v 4N rsavusu z dv uoko Lang Ldosd EMAIN GSddNI Lv DINSO 1 vc c ce Le 02 61 81 Zt 91 SL vL 51 0r 6 8 L 9 S v L JL 1 JL IL Aue pue Sjojepeuu SJojdeoei SJo19eJ pejeJoes seubo 81044 1eS onN ejo o 1 sesejeudsoug pue seseejpnu seseajoJd s sedseo oneuroqos e3aiy stsoidody gt Mo cm 2 n lt m O Q u u Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Apoptosis Profiler Gene List CONTROLS Coordinates Genbank beta actin M24 N24 O24 P24 X00351 J00074 M10278 Ubiquitin K24 L24 P7 15 amp 16 M26880 The following gene list is ordered alphabetically for your convenience Gene Coord Genbank Gene nam
3. 5 min Then incubate on ice for 5 min 2 Cutopen hybridization pouch add each DIG labeled cDNA probe directly to the Hybridization Buffer Step C 4 reseal pouch and hybridize over night 12 hours at 52 C 3 The next day prepare SSC SDS washing solutions as described in the Ap pendix Preheat SSC SDS solutions to 52 C before use Ensure that the SSC SDS solutions are free from all precipitates Shake well before use 4 Cut open hybridization pouch decant cDNA probe buffer and place mem brane into a clear plastic container such as a 200 Ul pipet tip container Quicky tinse each membrane with 10 ml 2X SSC 0 5 SDS and decant Wash twice with 30 ml of prewarmed 2X SSC 0 5 SDS at 52 C for 10 min per wash Then wash twice using 30 ml of prewarmed 0 1X SSC 0 5 SDS at 52 C for 10 min per wash Keep SSC SDS solutions at 52 C during wash steps Detection Note that all incubation and wash steps below should be performed on a rotat ing platform with moderate shaking at room temp 1 Blocking Decant 0 1X SSC 0 5 SDS washing solution and add 30 ml of 1X Blocking Buffer to each container 2 Incubate at room temperature for 30 min 3 Prepare anti DIG AP Antibody Mixture For each membrane mix 1 ml of 1X Blocking Buffer and 6 of anti DIG AP Antibody Mix well Add the 1 ml mixture to each container and incubate at room temperature for 30 min Note Be sure to spin the antibody conjugate for 5 minutes at maximum speed before
4. Ag L9 J02400 Simian virus 40 Large T Antigen mad M9 L06895 Human antagonizer of myc transcriptional activity MADD G20 NM 003682 Homo sapiens MAP kinase activating death domain MADD mRNA 4 20 U19727 Human microtubule associated protein 4 MAP4 mRNA complete cds MAPKAPK2 H3 NM_004759 Homo sapiens mitogen activated protein kinase activated protein kinase 2 MAPKAPK2 transcript variant 1 mRNA 5 13 XM 027021 Homo sapiens mitogen activated protein kinase activated protein kinase 5 MAPKAPK5 mRNA max N9 X60287 Human max gene MCL1 24 _ 021960 Homo sapiens myeloid cell leukemia sequence 1 BCL2 related MCL1 mRNA MDM2 120 7 12020 Human p53 associated gene MEK1 J3 L11284 Human activator kinase MEK1 gene MEK2 K3 111285 Homosapiens ERK activator kinase MEK2 mRNA MEK3 L3 NM 002756 Homo sapiens mitogen activated protein kinase kinase 3 MAP2K3 5 M3 NM 002757 Homo sapiens mitogen activated protein kinase kinase 5 MAP2K5 MEK6 N3 U39657 Human MAP kinase kinase 6 MKK6 mRNA MEKK1 AAC97073 MEK kinase 1 MEKK2 P3 NM_006609 Homo sapiens mitogen activated protein kinase kinase kinase 2 MAP3K2 Mrna MEKK3 A4 U78876 Human MEK kinase 3 gene 4 4 NM 005922 Homo sapiens mitogen activated protein kinase kinase kinase 4 transcript variant 1 mRNA 5 ASK1 C4 NM 005923 Homo sapiens mitogen activated protein kinase kinase kinase 5 MAP3K5 mRNA MKK
5. Homo sapiens tissue inhibitor of metalloproteinase 1 erythroid potentiating activity collagenase inhibitor TIMP1 mRNA TNF N18 X01394 Human tumor necrosis factor TNF R1 O18 X55313 Human TNF R gene for tumor necrosis factor receptor type 1 TNF R2 P18 U52156 Human tumor necrosis factor receptor 2 TNFR2 gene TNFAIP3 P20 NM_006290 Homo sapiens tumor necrosis factor alpha induced protein 3 TNFAIP5 mRNA TPO O17 NM_000460 Human thrombopoietin myeloproliferative leukemia virus oncogene ligand megakaryocyte growth and development factor THPO gene nur77 O11 L13740 Human TR3 orphan receptor mRNA complete cds TRADD 121 141690 Homo sapiens TNF receptor 1 associated protein TRADD mRNA TRAF1 JA NM 005658 Homo sapiens TNF receptor associated factor 1 TRAF1 mRNA TRAF3 CRAF1 21 NM_003300 Homo sapiens TNF receptor associated factor 3 TRAF3 mRNA TRAF4 L21 NM 004295 Homo sapiens TNF receptor associated factor 4 TRAF4 mRNA TRAF5 M21 AB000509 Homo sapiens mRNA for TRAF5 complete cds TRAF6 N21 NM 004620 Homo sapiens TNF receptor associated factor 6 TRAF6 mRNA TRAIL P19 U37518 Human TNF related apoptosis inducing ligand TRAIL gene TRAIL R1 0 149 U90875 Human cytotoxic ligand TRAIL receptor mRNA complete cds 2 DR5 M19 AF012535 Homo sapiens death receptor 5 DR5 mRNA complete cds TRAIL R3 DCR1 N19 AF020502 Homo sapiens cytotoxic TRAIL receptor 3 TRAIL R3 mRNA complete cds
6. M64673 Human heat shock factor 1 TCF5 mRNA complete cds HsRad51 A9 D13804 Human mRNA for RecA like protein HsRad51 complete cds I FLICE FLIP 123 AF041462 Homo sapiens I FLICE isoform 5 mRNA complete cds ICSBP1 9 NM 002163 Homo sapiens interferon consensus sequence binding protein 1 ICSBP1 mRNA IFN a G12 NM_002171 Homo sapiens interferon alpha 10 IENA10 mRNA IFN b J12 M35591 Human fibroblast interferon IFN beta gene IFN g K12 J00219 Human immune interferon IFN gamma gene IFNAR1 H12 NM_000629 Homo sapiens interferon alpha beta and omega receptor 1 IFNAR1 mRNA IFNAR2 112 NM_000874 Homo sapiens interferon alpha beta and omega receptor 2 IFNAR2 mRNA IFNGR1 L12 NM 000416 Homo sapiens interferon gamma receptor 1 IFNGR1 mRNA IFNGR2 M12 NM 005534 Homo sapiens interferon gamma receptor 2 interferon gamma transducer 1 2 mRNA IGF 1 N12 M29644 Human insulin like growth factor I gene IGF 2 O12 M29645 Human insulin like growth factor II gene IGFBP1 P12 M31145 Human insulin like growth factor binding protein mRNA complete cds Ik A13 NM 006083 Homo sapiens IK cytokine down regulator of HLA II IK mRNA IkB MAD3 c9 M69043 Human MAD 3 mRNA encoding IkB like activity IKK a N2 AF080157 Homo sapiens IkB kinase a IKK alpha mRNA IKK b 02 AF080158 Homo sapiens IkB kinase b IKK beta mRNA IKK g P2 AF074382 Homo sapiens IkB kinase gamma subunit IKK gamma mRNA IL 10 B13 U16720 Human
7. TRAIL R4 DCR2 O19 AF029761 Homo sapiens decoy receptor 2 mRNA complete cds TRIP O21 NM 005879 Homo sapiens TRAF interacting protein TRIP mRNA TRPM 2 P21 M64722 Human TRPM 2 mRNA complete cds VEGF P17 E14233 Human mRNA for vascular endothelial growth factor gene WAF1 11 U03106 Human wild type p53 activated fragment 1 WAF1 mRNA complete cds Wee 1 P6 NM 003390 Human weel S pombe homolog WEE1 mRNA Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Trademark Profiler is a trademark of Genemed Biotechnologies Inc CDP Svar is a registered trademark of Tropix Inc WARRANTY GENEMED BIOTECHNOLOGIES INC WARRANTS THAT THE MATERIALS SOLD MEET GENEMED S PERFORMANCE SPECI FICATIONS FROM THE TIME OF SHIPMENT UNTIL THE EXPIRATION DATE IF STORED UNDER THE RECOMMENDED CON DITIONS NO OTHER WARRANTIES OR GUARANTEES EXPRESSED OR IMPLIED ARE PROVIDED INCLUDING WARRANTIES FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE UNDER NO CIRCUMSTANCES SHALL GENEMED BE LIABLE FOR ANY DAMAGES ARISING OUT OF THE USE OF THE MATERIALS 2000 Genemed Biotechnologies Inc Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com gt rw
8. sapiens insulin receptor INSR mRNA IRAP IL 1RP 116 53296 H sapiens mRNA for IRF1 D9 NM_002198 Homo sapiens interferon regulatory factor 1 IRFI mRNA Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Gene Coord Genbank Gene name IRF2 E9 NM_002199 Homo sapiens interferon regulatory factor 2 IRF2 mRNA IRF4 F9 U52682 Human lymphocyte specific interferon regulatory factor interferon regulatory factor 4 LSIRF IRF4 mRNA B3 M64174 Human protein tyrosine kinase JAK1 gene JAK2 C3 NM_004972 Human Janus kinase 2 a protein tyrosine kinase JAK2 gene JAK3 D3 U09607 Human JAK family protein tyrosine kinase JAK3 gene JNK2 E3 NM_002752 Homo sapiens mitogen activated protein kinase 9 MAPK9 mRNA JNK3A1 F3 U34820 Human JNK3 alphal protein kinase JWK3A1 mRNA complete cds JNK3A2 G3 U34819 Human JNK3 alpha2 protein kinase JINK3A2 mRNA complete cds JunB G9 X51345 Human jun B mRNA for JUN B protein JunD H9 X51346 Human jun D mRNA for JUN D protein KIT M16 NM 000222 Homo sapiens v kit Hardy Zuckerman 4 feline sarcoma viral oncogene homolog mRNA L myc I9 M19720 Human L myc protein gene Lamin A J9 NM 005572 Homo sapiens lamin A C LMNA mRNA Lamin B1 K9 NM 005573 Homo sapiens lamin B1 LMNB1 mRNA Large T
9. 0110 Fax 650 952 1060 Email info genemed com www genemed com Gene Coord Genbank Gene name EPO H11 NM 000799 Human erythropoietin EPO EPOR I11 NM 000121 Human erythropoietin receptor EPOR MAPK1 F2 XM 009860 Homo sapiens mitogen activated protein kinase 1 MAPK1 mRNA Ets M8 J04101 Human erythroblastosis virus oncogene homolog 1 ets 1 mRNA Ets2 8 NM 005239 Homo sapiens v ets avian erythroblastosis virus E26 oncogene homolog 2 ETS2 mRNA FADD J19 NM 003824 Human Fas INFRSF6 associated via death domain FADD FAK G2 L13616 Human focal adhesion kinase FAK mRNA FAP H2 NM 004460 Homo sapiens fibroblast activation protein alpha FAP mRNA Fas E18 M67454 Human Fas Antigen Fas ligand F18 D38122 Human Fas ligand gene FGFR C12 M34185 Human fibroblast growth factor receptor FGFr transmem brane form FGF 1 Jii E03692 cDNA encoding human acidic FGF FGF 2 M27968 Human basic fibroblast growth factor FGF gene FGF 3 A12 NM 005247 Human fibroblast growth factor 3 murine mammary tumor virus integration site v int 2 oncogene homolog FGF3 gene FGF 4 B12 NM_002008 Human fibroblast growth factor 4 heparin secretory transforming protein 1 Kaposi sarcoma oncogene FGF4 gene FLT 1 D12 AF063657 Homo sapiens vascular endothelial growth factor receptor FLT1 Fodrina K19 XM 005369 Homo sapiens spectrin alpha non erythrocytic 1 alpha fodrin SPTAN1 mRNA Fodrinb A20 NM_003128 Hom
10. 4 D4 U17743 Human JNK activating kinase NKK1 mRNA 2 DUSP4 E4 U48807 Human MAP kinase phosphatase MKP 2 mRNA complete cds MLK F4 AF238255 Homo sapiens mixed lineage kinase mRNA complete cds MMP1 M23 U41078 Human membrane type matrix metalloproteinase 1 mRNA MNK2 G4 AF125532 Homo sapiens map kinase interacting kinase mRNA complete cds Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Gene Coord Genbank Gene name Myt1 H4 U56816 Human kinase Myt1 Myt1 mRNA complete cds n myc O9 Y00664 Human germ line n myc gene NAIP BIRC1 N23 U19251 Homo sapiens neuronal apoptosis inhibitory protein mRNA complete cds NFAT 1b P9 U43341 Human transcription factor NFAT1 isoform B NFAT mRNA 4 A10 L41067 Homo sapiens NF AT4c mRNA NFAT5 B10 AF134870 Homo sapiens transcription factor NFAT5 mRNA 1 C10 NM_003998 Homo sapiens nuclear factor of kappa light polypeptide gene enhancer in B cells 1 p105 NFKB1 mRNA 2 0109 61498 H sapiens mRNA for subunit NGF 2 G18 X53655 H sapiens mRNA for NGF 2 NGF b H18 NM_002506 Human nerve growth factor beta polypeptide NGFB gene NGFR L18 M14764 Human nerve growth factor receptor mRNA NIK I4 Y10256 H sapiens mRNA for setine threonine protein kinase NuMa E10 Z11583 H sapiens mRNA for NuMA protein O
11. Bax alpha gene BBP53 TP53BP2 C8 NM_005426 Homo sapiens tumor protein p53 binding protein 2 TP53BP2 mRNA bcl 6 E23 U00115 Human zinc finger protein bcl 6 mRNA complete cds Bel xL G23 Z23115 Human bcl xL gene Bcl2 F23 M14745 Human bcl 2 mRNA Bcl2 b H23 M13995 Human B cell proto oncogene Bcl2 w 123 U59747 Human Bcl w bcl w mRNA Bfl 1 J23 U27467 Human Bcl 2 related Bfl 1 mRNA complete cds BHRF1 A24 AF120456 Herpesvirus papio Bcl 2 homolog BHRF1 gene complete cds BID B24 NM_001196 Homo sapiens BH3 interacting domain death agonist BID mRNA C24 U34584 Human Bcl 2 interacting killer BIK mRNA BimEL D24 AF032457 Homo sapiens BimEL mRNA BIRC6 D21 NM_016252 Homo sapiens baculoviral IAP repeat containing 6 BIRC6 mRNA BIRC7 E21 NM_022161 Homo sapiens livin inhibitor of apotosis LIVIN mRNA BNIP 1 E24 NM_001205 Human BCL2 adenovirus E1B 19kD interacting protein 1 BNIP1 BNIP 2 F24 NM_004330 Human BCL2 adenovirus E1B 19kD interacting protein 2 BNIP2 Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Gene Coord Genbank Gene name BNIP 3 G24 NM 004052 Human BCL2 adenovirus E1B 19kD interacting protein 3 BNIP3 BOK H24 AF089746 Homo sapiens 1 2 related ovarian killer mRNA complete cds Bub1 AF053305 Human mitotic checkpoi
12. Human apoptosis related cysteine protease Caspase 3 C22 NM 004346 Human apoptosis related cysteine protease Caspase 4 D22 NM 001225 Human apoptosis related cysteine protease Caspase 5 E22 NM 004347 Human apoptosis related cysteine protease Caspase 6 F22 NM 001226 Human apoptosis related cysteine protease Caspase 7 G22 NM 001227 Human apoptosis related cysteine protease Caspase 8 H22 NM 001228 Human apoptosis related cysteine protease Caspase 9 122 NM 001229 Human apoptosis related cysteine protease CBL c cbl C19 X57110 Human c cbl proto oncogene CBL B D19 U26710 Human cbl b mRNA CD27 E17 M63928 Homo sapiens T cell activation antigen CD27 mRNA complete cds CD27 Ligand F17 L08096 Human CD27 ligand mRNA complete cds CD30 G17 M83554 H sapiens lymphocyte activation antigen CD30 mRNA complete cds CD30 Ligand A18 109753 Homo sapiens CD30 ligand mRNA complete cds CD4 B11 NM_000616 Homo sapiens CD4 antigen p55 CD4 mRNA CD40 B18 XM_009624 Homo sapiens tumor necrosis factor receptor superfamily member 5 5 mRNA Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Gene Coord Genbank Gene name CD40 Ligand C18 L07414 Human CD40 ligand mRNA complete cds CD45 O1 Y00062 Human mRNA for 200 leukocyte common antigen CD45 LC A CDC2 C6 X05360 H
13. PG 118 U94332 Human osteoprotegerin OPG mRNA complete cds OX40 J18 X75962 H sapiens mRNA for OX40 homologue OX40 Ligand K18 X79929 H sapiens OX40 ligand gp34 15 H7 L36844 Human clone p15INK4B HA5 CDK inhibitory protein ene plo I7 L27211 Puman CDK4 inhibitor p16 INK4 gene p18 J7 U17074 Human CDK6 inhibitor p18 gene 19 K7 U40343 Human CDK inhibitor p19INK4d gene p27 L7 X84849 Human p27kip1 gene p53 F10 103199 Human p53 Tumor Antigen p57 7 048869 Human cdk inhibitor p57 KIP2 gene p70 S6K SRK J4 AB019245 Human p70 S6Kb S6 kinase b gene PA2G4 N7 NM 006191 Homo sapiens proliferation associated 2G4 38kD PA2G4 mRNA 1 K4 NM_002576 Homo sapiens p21 Cdc42 Racl activated kinase 1 yeast Ste20 related PAK1 mRNA PARP G10 NM 001618 Human ADP ribosyltransferase NAD poly ADP ribose polymerase ADPRT gene PCNA O7 NM 002592 Human proliferating cell nuclear antigen PCNA gene PDGF R H17 03278 Human platelet derived growth factor PDGF receptor mRNA PDGF a N16 X06374 Human mRNA fot platelet derived growth factor PDGF A PDGF b O16 X02811 Human platelet derived growth factor B chain PDGF B gene perforin 023 M28393 Human perforin mRNA complete cds 1 I4 NM 012395 Homo sapiens PFTAIRE protein kinase 1 PFTK1 mRNA PI3 Kinase M4 Y11312 Human gene for phosphoinositide 3 kinase Pitslare A1 4 U04815 Human protein kinase PITSLRE alpha 1 mRNA PKA a O4 X07767 Human mRNA for cAMP dep
14. array membranes are washed and subsequently in cubated with Anti DIG Alkaline Phosphatase DIG AP Antibody Upon addition of the CDP Star chemiluminescent substrate provided detection of DIG AP can be obtained within minutes by exposing array membranes to film or by using a chemiluminescent imaging system such as Alpha Innotech s FluorChem Imaging System Page 4 Genemed Biotechnologies Inc Profiler Array Nonradioactive System II PACKAGE CONTENTS the front cover of this User Manual for appropriate storage conditions For Buffer Solutions amp Recipes see Appendix at the back of this User Manual Profiler Array Nonradioactive System Profiler Array and Hybridization Kit Store at room temp 2 Profiler Arrays 25ml Hybridization Buffer 60ml 20X SSC 60ml 10 SDS Array cDNA Synthesis Kit Store at 20 C 10 ug Control K562 Total RNA yellow e 10 Reverse Transcriptase amber 15u Random Primer Mix blue 15u Probe pink e 50u 100mM DTT salmon e 100u 5X RT Buffer purple e 100 dH 0 natural Profiler Array Chemiluminescence Detection Kit Store at 4 C 15ml CDP Svzar Substrate 30ml 5X Blocking Buffer 50ml 10X Antibody Wash Buffer I 50ml 10X Antibody Wash Buffer II 30ml 10X Detection Buffer e 15 Alk Phosphatase Conjugate orange Additional Materials Provided 1 Profiler Grid 1 Product User Manual Tsee Product Analysis C
15. ch as Alpha Innotech s FluorChem or equivalent Be sute to read Section G below to find out how to normalize the genes on the arrays For a quick cutsory analysis of results you may perform the following 1 Place the two x ray images together such that the housekeeping genes are aligned 2 Slowly shift one x ray film to the left or right and observe any differences in spot intensity Spots that exhibit differences in signal intensity may indi cate that a gene is upregulated or downregulated however results are not conclusive until you have normalized the genes on the arrays Normalization On each array two housekeeping genes beta actin and ubiquitin have been spotted at different locations on the array Four beta actin spots have been placed on the lower right hand corner for orientation purposes see Appendix Select one housekeeping gene On each array take the average intensity for the housekeeping gene of your choice and use this value as the baseline Normalize each spot with respect to the average value of the housekeeping gene After normalizing each array compare spot intensities on separate arrays for each gene Generally a threefold increase or decrease in spot intensity is considered significant Genemed Biotechnologies Inc Page9 Profiler Array Nonradioactive System V TROUBLESHOOTING GUIDE The following list of tips is designed to help you troubleshoot problems that you may encounter when performin
16. clear factor kappa B ligand RANKL mRNA complete cds rasGAP K20 D78155 Homo sapiens mRNA for rasGTPase activating protein complete cds Rb 110 J10 M28419 Human retinoblastoma susceptibility protein mRNA Rb2 p130 K10 X74594 Human Rb2 p130 protein RelA L10 L19067 Human NF kappa B transcription factor p65 subunit mRNA RelB M10 NM 006509 Human v rel avian reticuloendotheliosis viral oncogene homolog B nuclear factor of kappa light polypeptide gene enhancer in B cells 3 RELB gene RIP3 JS AF156884 Homo sapiens RIP like kinase RIP3 mRNA complete cds RIPK1 K5 NM_003804 Homo sapiens receptor TNFRSF interacting serine threonine kinase 1 RIPK1 mRNA RIPK2 L5 NM 003821 Homo sapiens receptor interacting serine threonine kinase 2 RIPK2 mRNA RPS6KA6 M5 AF184965 Homo sapiens ribosomal S6 kinase RPS6KA6 mRNA complete cds SAPK4 N5 AF004709 Homo sapiens stress activated protein kinase 4 mRNA complete cds SCF 117 M59964 Human stem cell factor mRNA Sentrin L20 U83117 Human sentrin mRNA complete cds SOD1 M20 X02317 Human mRNA for Cu Zn superoxide dismutase SOD SPHK1 O5 NM 021972 Homo sapiens sphingosine kinase 1 SPHK1 mRNA SPHK2 5 AF245447 Homo sapiens sphingosine kinase type 2 isoform mRNA complete cds Statl N10 M97935 Homo sapiens transcription factor ISGF 3 mRNA Stat2 O10 NM 005419 Homo sapiens signal transducer and activator of transcription 2 113kD STAT2 mRNA Stat3 P10 AJ012463 Homo sapiens mRNA f
17. diluting in 1X Blocking Buffer Some lots may contain aggregated conjugates that precipi tate and can cause a spotty background on the membrane 4 Wash the membranes at room temperature Remove the anti DIG AP Antibody Mixture Blocking Buffer and wash twice using 40 ml of 1X An tibody Wash Buffer I for 10 min per wash Then repeat washes using 40 ml of 1X Antibody Wash Buffer II for 10 min per wash 5 Dilute CDP Star Substrate in 1X Detection Buffer 1 30 for x ray film or 1 10 for Imager Incubate each membrane in 30 ml diluted substrate for 30 minutes at room temperature 6 Using forceps remove each membrane from the diluted substrate Do not allow the membrane to dry We recommend placing the membrane on a plastic support such as a developed used x ray film Page 8 Genemed Biotechnologies Inc IV Profiler Array Nonradioactive System INSTRUCTIONS FOR USE continued 7 Immediately cover each membrane with plastic wrap e g Saran Wrap or plastic sheet protectors and remove excess substrate Avoid air bubbles Expose the membrane to Hyperfilm ECL Amersham Cat RPN31144 for 3 10 min at room temperature We suggest trying several different ex posures of varying lengths of time 1 2 10 min Alternatively use a chemi luminescent imaging system such as Alpha Innotech s FluorChem Imager ot equivalent Interpretation of results For quantitative results we suggest using an chemiluminescent imaging system su
18. e 4 1BB A17 UO3397 Human receptor protein 4 IBB mRNA complete cds 4 1BB Ligand B17 U03398 Human receptor 4 1BB ligand mRNA complete cds ACP5 B1 NM 001611 Homo sapiens acid phosphatase 5 tartrate resistant ACP5 mRNA C1 NM 005163 Human v akt murine thymoma viral oncogene homolog 1 AKT1 Akt2 D1 NM_001626 Homo sapiens v akt murine thymoma viral oncogene homolog 2 2 A8 NM 003220 Human transcription factor AP 2 alpha activating enhancer binding protein 2 alpha TFAP2A gene APAF 1 A21 AF013263 Human apoptotic protease activating factor 1 Apaf 1 gene APIA BIRC5 B21 _008623 Homo sapiens baculoviral repeat containing 5 survivin BIRC5 mRNA Apo 3 CIT U74611 Human Apo 3 mRNA complete cds Apo 3 Ligand D17 AF055872 Homo sapiens Apo3 DR3 ligand APO3L mRNA complete TWEAK cds ARC C21 AF043244 Homo sapiens apoptosis repressor ARC mRNA complete cds 19 AB023416 Homo sapiens ASC mRNA for apoptosis associated speck like protein containing a CARD complete cds ATM E1 NM 000051 Homo sapiens ataxia telangiectasia mutated includes complementation groups A C and D ATM mRNA b catenin B19 Z19054 Human beta catenin gene b myb B8 X13293 Human B myb oncogene Bad A23 AF031523 Human bcl xL bcl 2 associated death promoter BAD gene 1 23 _ 004323 Homo sapiens BCL2 associated athanogene BAG1 mRNA C23 U16811 Human Bak mRNA Bax a D23 L22473 Human
19. e hybridization time An overnight hybridization 12 hours is necessary for optimal binding of the probe to the array How much CDP Star substrate did you use Make sure that the substrate is evenly distributed over the membrane during detection Step E5 e How long was the exposure Try several exposures for varying lengths of time This is necessary for ob taining optimal results Page 10 Genemed Biotechnologies Inc INOL LOS 53005 unoe eyeg Profiler Array Nonradioactive System APPENDIX Array Orientation VI Page11 Genemed Biotechnologies Inc VI APPENDIX continued Solution Dilution Recipes enough for two array membranes 250 ml of 2X SSC 0 5 SDS To 212 5 ml of deionized H2O add 25 ml of 20X SSC stock solution and 12 5 ml of 10 SDS stock solution Shake well before use 250 ml of 0 1X SSC 0 5 SDS To 236 25 ml of deionized H2O add 1 25 ml of 20X SSC stock solution and 12 5 ml of 10 SDS stock solution Shake well before use 75 ml of 1X Blocking Buffer To 60 ml of deionized H2O add 15 ml of 5X Blocking Buffer stock solution Shake well before use 200 ml of 1X Antibody Wash Buffer I To 180 ml of deionized H2O add 20 ml of 10X Antibody Wash Buffer I stock solu tion Shake well before use 200 ml of 1X Antibody Wash Buffer II To 180 ml of deionized H2O add 20 ml of 10X Antibody Wash Buffer II stock solution Shake well before use 100 ml of 1X Detection Buffer To 90 ml of deioni
20. endent protein kinase catalytic subunit type alpha PKC a P4 X52479 Human PKC alpha protein kinase C alpha gene PKC b1 A5 X06318 Human gene protein kinase type beta 1 PKC delta B5 NM_006254 Homo sapiens protein kinase C delta PRKCD mRNA PKC r C5 Z15114 H sapiens gene for protein kinase C gamma PLK D5 NM_005030 Homo sapiens polo Drosophia like kinase PLK mRNA PP2A cs JS XM_003944 Homo sapiens protein phosphatase 2 formerly 2A catalytic subunit alpha isoform PPP2CA mRNA PPPICB E5 NM 002709 Homo sapiens protein phosphatase 1 catalytic subunit beta isoform PPP2R5A G5 XM 010692 Homo sapiens protein phosphatase 2 regulatoty subunit B B56 alpha isoform PPP2R5A mRNA Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Gene Coord Genbank Gene name presenilin 1 J20 L76517 Homo sapiens clone 44 senilin 1 PS1 182 mRNA complete cds PU 1 SPI1 H10 X52056 Human mRNA for spi 1 proto oncogene PYK2 FAK2 H5 U33284 Human protein tyrosine kinase PYK2 mRNA complete cds Raf A Raf 1 1 X04790 Human mRNA for A raf 1 oncogene Raf c rafl I5 X03484 Human mRNA for raf oncogene RAN 110 NM 006325 RAN member RAS oncogene familyHomo sapiens RAN member RAS oncogene family RAN mRNA RANKL TRANCE M18 AF019047 Homo sapiens receptor activator of nu
21. eptor beta ILZRB mRNA IL 2R g H15 NM 000206 Homo sapiens interleukin 2 receptor gamma severe combined immunodeficiency ILZRG mRNA IL 2Ra F15 NM 000417 Homo sapiens interleukin 2 receptor alpha IL2RA mRNA IL 3 I15 M14743 Human interleukin 3 IL 3 IL 3Ra J15 M74782 Human interleukin 3 receptor hIL 3Ra mRNA IL 4 K15 NM 000589 Human interleukin 4 IL4 IL 4Ra L15 NM 000418 Homo sapiens interleukin 4 receptor ILAR mRNA IL 5 M15 J03478 Human interleukin 5 IL 5 gene IL 5Ra N15 M96652 Human interleukin 5 receptor alpha subunit IL5R mRNA complete cds IL 6 O15 M54894 Human interleukin 6 mRNA IL 6R A16 X12830 Human mRNA for interleukin 6 IL 6 receptor IL 6ST B16 NM_002184 Homo sapiens interleukin 6 signal transducer gp130 oncostatin M receptor IL6ST mRNA IL 7 C16 j04156 Human interleukin 7 IL 7 IL 7R D16 M29696 Human interleukin 7 receptor IL 7 mRNA IL 8 E16 M28130 Human interleukin 8 IL8 gene IL 8R F16 M68932 Human IL 8 receptor mRNA IL 8Rb G16 NM 001557 Homo sapiens interleukin 8 receptor beta ILBRB mRNA IL 9 H16 XM_003760 Homo sapiens interleukin 9 IL9 mRNA IL 9R 116 NM 002186 Homo sapiens interleukin 9 receptor mRNA iNOS F20 L09210 Homo sapiens inducible nitric oxide synthase mRNA complete cds INPP5D SHIP A3 NM 005541 Homo sapiens inositol polyphosphate 5 phosphatase 145kD INPP5D mRNA Insulin J16 J00265 Human insulin gene Insulin Receptor K16 XM_048346 Homo
22. ertificate provided with each kit for complete list of genes spotted on the array Genemed Biotechnologies Inc Page 5 Profiler Array Nonradioactive System ADDITIONAL MATERIALS amp EQUIPMENT Additional Materials Required Sheared salmon testes DNA Sigma Cat D7656 Hyperfilm ECL Film Amersham Cat RPN31144 Plastic wrap e g Saran Wrap or plastic sheet protectors Commercial pouch sealer or equivalent Additional Materials Recommended Trizol Life Technologies Cat 15596 018 or equivalent Additional Equipment IV Thermal cycler or water bath Temperature controlled Shaking or Rotating platform Optional Hybridization oven and bottles Stratagene Cat 401031 Optional Alpha Innotech FluorChem or equivalent INSTRUCTIONS FOR USE Isolation of total RNA from cells or tissues Total RNA can be isolated from tissues or cells using standard methods e g Trizol or equivalent You can use poly A RNA if you wish however we have found that equivalent results can be achieved using total RNA Quantify your RNA samples by measuring the absorbance at 260 nm and 280 nm Total RNA samples that are free of proteins have an Ay ratio of 1 8 2 2 In addition you should check the quality of your RNA by agarose gel electrophoresis Run 0 5 2 0 ug of RNA on a denaturing formaldehyde agar ose ethidium bromide gel Two bright bands 285 and 185 rRNA should be observed at approximately 5 and 2 kb respective
23. g the procedure If you feel you need additional technical assistance please contact us by phone fax or email A Problem high background Was tbe membrane completely immersed in solution during the incubations Never let the membrane dry out and be sure membrane is completely im mersed during all incubations If desired paper clips can be used to clip mem branes to the bottom of the plastic containment during washing blocking detection steps Use extreme caution when using this method Was the membrane completely covered in the solutions during detection Be sure solutions completely cover the membrane during these steps Were the hybridization solutions completely dissolved Check Hybridization Buffer for visible precipitated particles 5 the film overexposed Try several exposures for varying lengths of time This is necessary for ob taining optimal results eHow much antibody did you add to the Blocking Buffer in Step E3 Make sure the antibody is diluted 1 5 000 Problem hybridization signals weak or absent Is tbe problem witb your RNA quality Be sure to check the quality of your RNA Section IV A eAre the kit reagents working properly Strip the membrane and use the control K562 RNA provided to confirm that reagents and materials are working propetly e Did you hybridize the membrane at the correct temperature Check to make sure the hybridization temperature is set at 52 C e How long was th
24. interleukin 10 IL10 gene IL 10Ra C13 NM 001558 Homo sapiens interleukin 10 receptor alpha mRNA IL 10Rb D13 NM 000628 Homo sapiens interleukin 10 receptor beta IL10RB mRNA IL 11 E13 M57765 Human interleukin 11 mRNA IL 11RA F13 NM_004512 Homo sapiens interleukin 11 receptor alpha IL11RA mRNA IL 12 p35 G13 AF180562 Homo sapiens interleukin 12 P35 mRNA IL 12 p40 H13 AF180563 Homo sapiens interleukin 12 P40 gene IL 12RB1 113 NM 005535 Homo sapiens interleukin 12 receptor beta 1 ILI2RBI mRNA IL 12RB2 J13 NM 001559 Homo sapiens interleukin 12 receptor beta 2 IL12RB2 mRNA IL 13 K13 L06801 Homo sapiens interleukin 13 mRNA complete cds IL 13RA1 L13 NM 001560 Homo sapiens interleukin 13 receptor alpha 1 IL13RA1 mRNA IL 13RA2 M13 NM 000640 Homo sapiens interleukin 13 receptor alpha 2 IL13RA2 mRNA IL 14 N13 L15344 Human high molecular weight B cell growth factor mRNA sequence IL 15 O13 U14407 Human interleukin 15 IL15 mRNA complete cds IL 15RA P13 NM 002189 Homo sapiens interleukin 15 receptor alpha IL15RA mRNA IL 16 A14 NM 004513 Homo sapiens interleukin 16 lymphocyte chemoattractant factor IL16 mRNA Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Gene Coord Genbank Gene name IL 17 B14 U32659 Human IL 17 mRNA comple
25. l CCNA1 mRNA Cyclin B1 7 25753 Human cyclin mRNA 3 Cyclin 7 NM_005190 Human cyclin C CCNC gene Cyclin D1 C7 M64349 Human cyclin D1 Cyclin E D7 M73812 Human cyclin E mRNA sequence Cyclin F E7 NM 001761 Homo sapiens cyclin F CCNF mRNA Cyclin G1 F7 U53328 Human cyclin G Cyto C1 G19 NM_001916 Homo sapiens cytochrome c 1 CYC1 mRNA DAP Kinase B2 X76104 H sapiens DAP kinase mRNA DAXX H19 NM_001350 Homo sapiens death associated protein 6 DAXX mRNA DDX1 119 NM 004939 Homo sapiens DEAD H Asp Glu Ala Asp His box polypeptide 1 DDX1 DFFA K22 NM 004401 Homo sapiens DNA fragmentation factor 45 kD alpha polypeptide DFFA mRNA DFFB L22 NM 004402 Homo sapiens DNA fragmentation factor 40 kD beta polypeptide caspase activated DNase DFFB mRNA DNA PK C2 U47077 Homo sapiens DNA dependent protein kinase catalytic subunit DNA PKcs mRNA complete cds DR6 D18 AF068868 Homo sapiens TNFR related death receptor 6 DR6 mRNA complete cds DRAK1 D2 AB011420 Homo sapiens mRNA for DRAKI complete cds DRAK2 E2 AB011421 Homo sapiens mRNA for DRAK2 complete cds ElAc K8 M18288 Adenovirus type 40 E1A and E1B genes E2F 1 L8 M96577 Human E2F 1 pRB binding protein gene F11 NM_001963 Human epidermal growth factor EGF EGFR G11 NM 005228 Human epidermal growth factor receptor Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952
26. ll by pipetting Then incubate at 42 C for 2 hr After incubation samples at 75 C for 10 min to inactivate RT Proceed directly to Step D Hybridization or store DIG labeled cDNA probe s at 20 until use Pre Hybridization Before you start preheat Hybridization Buffer to 52 Completely dissolve all precipitates in the Hybridization Buffer Prepare sheared salmon testes DNA 1 Heat the sheared salmon testes DNA 10 mg ml at 905 100 C for 5 min then immediately place on ice 5 min We recommend preparing aliquots of 100 ul i e 50 per membrane then storing at 20 C until use Place each membrane into a hybridization pouch with the spots facing up Note the orientation of the membranes The correct orientation is shown in the Appendix indicating that the notch is on the top right hand corner Fill the pouch entirely with pre warmed 5 ml deionized H O avoid adding H O directly onto the membrane Then carefully decant water For each membrane prepare pre hybridization solution Add 50 of the sheared salmon testes DNA to 5ml of pre warmed Hybridization Buffer After mixing add 5 ml of pre hybridization solution to each pouch con taining the membrane Remove bubbles before sealing the pouch Incubate at 52 C for 22 hr Genemed Biotechnologies Inc Page7 Profiler Array Nonradioactive System IV D E INSTRUCTIONS FOR USE continued Hybridization 1 Heat DIG labeled cDNA probe at 94 C for
27. ly These bands are sharply defined and visible at about a 2 1 ratio 288 185 Preparation of cDNA probe For best results we recommend using a thermal cycler for all incubation steps however if a thermal cycler is unavailable heating blocks may be used If de sired first perform prehybridization Step C 1 4 and then prepare RNA Step B 2 and cDNA synthesis mixture Step B 4 simultaneously 1 Preheat a thermal cycler or heating block or equivalent to 70 Page 6 Genemed Biotechnologies Inc IV Profiler Array Nonradioactive System INSTRUCTION FOR USE continued 2 For each total RNA sample combine the following components into a sterile 0 5 ml microcentrifuge tube 5 10 Ug Total 3 Random Primer Mix Bring each tube to a final volume of 10 Hl by adding dH O provided Mix well by pipetting and spin briefly in a microcentrifuge Keep on ice Heat samples at 72 C for 2 min Then reduce temperature to 42 C and incubate for 1 2 min Note If a heating block is used tubes must be incubated for a minimum of 2 min at 42 C Prepare the cDNA synthesis mixture for each total RNA sample 6 5X First Strand Buffer 01MDTT 6 ul Probe Mix 1 Reverse Transcriptase 4H HO provided 20 Total volume per RNA sample Mix well by pipetting Incubate the cDNA synthesis mixture at 42 C for 2 min Transfer the mixture to the tube containing the RNA sample from Step B3 and mix we
28. m D oTa eneme Biotechnologies Inc PRODUCT ANALYSIS CERTIFICATE Apoptosis Profiler Array Nonradioactive System FOR RESEARCH USE ONLY CATALOG 05 102 SIZE 1 KIT Description The Apoptosis Profiler Array Nonradioactive System employs digoxigenin DIG 11 dUTP labeled cDNA and chemiluminescent detection Each Apoptosis Profiler Array contains 279 human genes which have been spotted onto a positively charged nylon membrane The spots on the array consists of popular and well studied genes that have been implicated in cell cycle regulation and apoptosis Enough reagents are provided for four hybridizations Package Contents Profiler Array and Hybridization Kit 2 Apoptosis Profiler Arrays 25 ml Hybridization Buffer 60ml 20X SSC 60ml 10 SDS Profiler Array cDNA Synthesis Kit 10 Control K562 Total RNA 10 Reverse Transcriptase 15 Random Primer Mix 15 Probe Mix 50 100mM DTT 100 SX RT Buffer 100 wh dH20 Profiler Array Chemiluminescence Detection Kit 15 ml CDP Svzar Substrate 15 ml Alk Phosphatase Conjugate 30 ml 5 Blocking Buffer 50ml 10X Antibody Wash Buffer I 50ml 10X Antibody Wash Buffer II 30ml 10X Detection Buffer Additional Materials 1 Profiler Grid 1 Product User Manual Storage Store the Profiler Array and Hybridization Kit at room temperature Profiler Array cDNA synthesis Kit at 20 C and Profiler Array Chemiluminescence Kit at 4 C Shelf Life 1 yea
29. nt kinase Bub1 BUB1 gene Bub3 B6 AF081496 Homo sapiens kinetochore protein BUB3 BUB3 mRNA c abl F1 NM 005157 Homo sapiens v abl Abelson mutine leukemia viral oncogene homolog 1 1 c fos D8 K00650 Human Proto oncogene c fyn G1 NM 002037 Human FYN oncogene related to SRC FGR YES FYN gene c jun E8 J04111 Human c jun proto oncogene c lck H1 NM 005356 Homo sapiens lymphocyte specific protein tyrosine kinase LCK c lyn H M16038 Human lyn mRNA encoding a tyrosine kinase c mpl All M90102 Human clones 15 39 41 c myeloproliferative leukemia virus type P c mpl P mRNA c myb F8 M15024 Human Proto oncogene c myc G8 E01841 Human proto oncogene c rel H8 NM 002908 Homo sapiens v rel avian reticuloendotheliosis viral oncogene homolog REL mRNA C stC JA X59932 Human C SRC kinase CAD F21 AB013918 Homo sapiens mRNA for CAD Calcineurin A1 K1 M29550 Human calcineurin 1 mRNA Calcineurin B LI M30773 Human calcineurin mRNA CAMK2B M1 NM 001220 Homo sapiens calcium calmodulin dependent protein kinase CaM kinase II beta CAMK2B mRNA CAMK2D 1 NM_001221 Homo sapiens calcium calmodulin dependent protein kinase CaM kinase delta CAMK2D mRNA Caspase 1 G21 NM 001223 Human apoptosis related cysteine protease Caspase 10 H21 NM 001230 Human apoptosis related cysteine protease Caspase 13 A22 AF078533 Homo sapiens evolutionarily related interleukin 1beta converting enzyme mRNA complete cds Caspase 2 B22 NM 001224
30. o sapiens spectrin beta non erythrocytic 1 SPTBN1 mRNA fos B O8 NM 006732 Homo sapiens FBJ murine osteosarcoma viral oncogene homolog B FOSB mRNA FRP 1 I2 U49844 Human FRAP related protein FRP1 mRNA GADD45 G7 M60974 Human growth arrest and DNA damage inducible protein gadd45 GCK J2 NM 000162 Homo sapiens glucokinase hexokinase 4 maturity onset diabetes of the young 2 GCK nuclear gene encoding mitochondrial protein mRNA GCSF E12 NM_000759 Human colony stimulating factor 3 granulocyte CSF3 GMCSF F12 NM 000758 Human colony stimulating factor 2 granulocyte macrophage CSF2 GPRK6 K2 XM 003736 Homo sapiens G protein coupled receptor kinase 6 mRNA GPX1 B20 M21304 Human glutathione peroxidase GPX1 mRNA complete cds Granzyme A M22 NM 006144 Homo sapiens granzyme granzyme 1 cytotoxic T lymphocyte associated serine esterase 3 GZMA mRNA Granzyme B N22 M28879 Human granzyme B CTLA 1 gene GSK3B L2 NM 002093 Homo sapiens glycogen synthase kinase 3 beta GSK3B mRNA GSTM1 C20 NM_000561 Homo sapiens glutathione S transferase M1 GSTM1 mRNA Hrk 124 U76376 Homo sapiens activator of apoptosis Hrk HRK mRNA complete cds Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Gene Coord Genbank Gene name HSF 1 5 E20
31. ogous regions among the genes on the array so there is no risk of crosshybridization As a result your labeled cDNA probe will hybridize effi ciently and selectively to specific sequences on the array In addition we offer targeted arrays such as the Oncogene Profiler Array and Apoptosis Profiler Array For a complete list of genes for each array see the Product Certificate Sheet provided with each kit Benefits of the Profiler Array Unlike other macroarrays which often require poly A RNA the Profiler Array has no preference for total or poly A RNA In fact our method provides equivalent results using either total RNA or mRNA so there is no need for special RNA purification procedures Genemed Biotechnologies Inc Page 3 Profiler Array Nonradioactive System I INTRODUCTION continued Sample 1 Sample 2 e g control e g experimental Obtain cell or tissue samples U Uu Isolate Total RNA s ei 1 6 hr AAAA NA Gv O al AAAA AAA Prepare Labeled cDNA amp be pa DIG DIG id Hybridization Bag Hybridize overnight 712 hr E ig gt US SE p 2 T 3 Detect amp analyze Rr C am be 3 4 hr 5 min exposure to x ray film Total Time 1 2 days 24 28 hr Figure 1 provides a simplified overview of the Profiler procedure After labeling Profiler Arrays are hybridized overnight with DIG 11 dUTP labeled cDNA The next day the
32. ons ules ia vat Jorxo 9491 v 3 di ANSI 2393 via eid LSSHO DidlH Divd 9Mud9 1 dvoseud uupoJ HION 14vHl 154 aava orxo 451 pye 01 11 giaz PNA 1 494 Onda y uwe gid expo HAIN 9 92 H 6 odo 40S uen 1 61 11 guzi WzH NdI HOd3 9 ur 18349 919 ZPO lavi MIN LdHd u j o g dseg 01 00 xxva 6 71 AYZ 1981 71 0 Oda 1 3389 Sid 2MAd dv dINg z dseg 1 dseo aav oof z4oN oeao 9 1 992 11 981 71 89 21 11 NAI YADA duvd gunf o amp u o seaavo SAPO vsHeddd ZANIN ZENE c amp dIN8 2 08 o AY SON edo P4871 PST PHLI TIIASOWD 493 Sd vui GAWD PAPO WOZddd FPEMNC Iqe 5 9199 9 dseS Z29819 ISH aavuo sea 1042 811 HZL M ASOD 1 450 WNN unto 4 xpo 89 ddd Z dMIN OIN WLY exeg dseo 90819 LISD 8799 9ua TeOdV ee adzi HLA H4INO z89JN kabi sor ZAPO bd PDA XVP bivHO ZDIV
33. or transcription factor Stat4 L11 L78440 Human STAT4 gene Stat5a M11 U43185 Human signal transducer and activator of transcription Stat5A gene Stat6 N11 _ 003153 Human signal transducer and activator of transcription 6 interleukin 4 induced STAT6 gene 1 20 U49928 Homo sapiens binding protein TAB1 mRNA complete cds MAP3K7 AF218074 Homo sapiens TGF beta activated kinase splice variant d TAK1 mRNA complete cds TANK TRAF2 020 U59863 Human TRAF interacting protein I TRAF mRNA complete cds TGF a J17 M31172 Human transforming growth factor alpha gene TGF b K17 X02812 Human transforming growth factor beta gene TGFb RI L17 NM 004612 Homo sapiens transforming growth factor beta receptor I activin A receptor type II like kinase 53kD TGFBR1 mRNA TGFb RII M17 NM 003242 Homo sapiens transforming growth factor beta receptor 70 80kD TGFBR2 mRNA Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com Gene Coord Genbank Gene name TGFb RIII N17 NM 003243 Homo sapiens transforming growth factor beta receptor betaglycan 300kD TGFBR3 mRNA TIE 1 AF023676 Homo sapiens lamin B receptor homolog TM7SF2 75 2 mRNA TIE 2 TERK O6 L06139 Homo sapiens receptor protein tyrosine kinase TEK mRNA complete cds TIMP 1 P23 NM_003254
34. otechnologies Inc Profiler Array Nonradioactive System I INTRODUCTION With the advent of macroarray technology studying differential gene expres sion has become relatively easy and routine The expression profiles of hun dreds of genes can be studied simultaneously in a single array experiment Typically array experiments require the use of radioactive cDNA probes However working with radioactively labeled probes is a hassle requiring time consuming procedures Furthermore radioactively labeled cDNA arrays tend to generate high levels of background The Profiler Array provides researchers with an alternative approach a hassle free nonradioactive array system that employs digoxigenin DIG 11 dUTP labeled cDNA and chemiluminescent detection With this method you can obtain semiquantitative results in just 1 2 days compared to 4 7 days that radioactive based methods demand Our first array the Star Profiler Array contains 373 genes an exclusive set of the most popular and well studied genes spotted onto a positively charged nylon membrane Star Profiler Array contains genes that play a significant role in biological functions and are corroborated with published literature thus a single experiment using Star Profiler Arrays generates highly informative data In addition each spot contains long oligonucleotides gt 90 nt synthesized from a carefully selected sequence Our selection process eliminates any ho mol
35. r from date of receipt under proper storage conditions Shipping Conditions Blue ice 4 C or room temperature Quality Control A sample of this kit was tested according to the user manual cDNA probes were prepared from Control K562 Total RNA PACO090 Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 877 GENEMED 436 3633 or 650 952 0110 Fax 650 952 1060 Email info genemed com www genemed com N ab m Z Q m m ono ve c be 0c 61 81 ZL 91 SL v 51 ZL LL OL 6 8 z 9 5 v L L JIM ZMHdS Oxd ZAAIN AYAL IL MSHO 080 lihas pan bi svao unoe ereg LAIL 2 dvi gu JN1 SOSA bon z Pas kdg9 I3vM E S qilV 4N OdNHuu uuoyad qviu ANYL JPY TIVYLLH ANL Odl amp 35ad 9 11 Tic 8171 2 491 939 vNOd bnoe ejeq dIvN E 1 gvl Jed vui ANL 491 9 OHS I H 0491 omms 145 xew 2 59 voevd Pavs Cex LANN ou cues LAOS Ru vui S M zur feses giey peu isa 199 UJON 09302 OUP LAT poser ERANT ps Bele m Jz3 zd AYL 9wi9SdH SHAW SOMWVO 8 Aldd EXIN 8 uuneurjeg v p
36. te cds IL 17B C14 NM 014443 Homo sapiens interleukin 17B IL17B mRNA IL 17BR D14 AF212365 Homo sapiens IL 17B receptor IL17BR mRNA complete cds IL 17R E14 NM 014339 Homo sapiens interleukin 17 receptor IL17R mRNA IL 18 F14 NM 001562 Homo sapiens interleukin 18 interferon gamma inducing factor 118 mRNA IL 18BP G14 NM 005699 Homo sapiens interleukin 18 binding protein IL18BP mRNA IL 18R1 H14 NM 003855 Homo sapiens interleukin 18 receptor 1 IL18R1 mRNA IL 19 I14 XM 001404 Homo sapiens interleukin 19 IL19 mRNA IL 1a 14 X03833 Human gene for interleukin 1 alpha IL 1 alpha IL 1b K14 M15330 Human interleukin 1 beta mRNA complete cds IL 1R1 L14 NM 000877 Homo sapiens interleukin 1 receptor type I ILIR1 mRNA IL 1R2 M14 NM 004633 Homo sapiens interleukin 1 receptor type II ILIRZ mRNA IL 1RL1 N14 NM_016232 Homo sapiens interleukin 1 receptor like 1 ILIRL1 mRNA IL 1RL2 O14 NM 003854 Homo sapiens interleukin 1 receptor like 2 ILIRL2 mRNA IL 2 P14 U25676 Human interleukin 2 TL 20 A15 NM_018724 Homo sapiens interleukin 20 IL20 mRNA IL 21 15 AF254069 Homo sapiens interleukin 21 IL21 mRNA complete cds IL 21R C15 AF254067 Homo sapiens interleukin 21 receptor IL21R mRNA complete cds IL 22 D15 NM_020525 Homo sapiens interleukin 22 IL22 mRNA IL 22R E15 AF286095 Homo sapiens IL 22 receptor IL22R mRNA complete cds IL 2R b G15 NM 000878 Homo sapiens interleukin 2 rec
37. uman CDC2 gene involved in cell cycle control Cdk2 D6 _001798 Human cyclin dependent kinase 2 CDK2 Cdk3 E6 NM 001258 Human cyclin dependent kinase 5 CDK3 Cdk4 F6 NM 000075 Human cyclin dependent kinase 4 CDK4 Cdk5 G6 NM 004935 Human cyclin dependent kinase 5 CDK5 Cdk6 H6 NM 001259 Human cyclin dependent kinase 6 CDK6 Cdk7 16 NM_001799 Homo sapiens cyclin dependent kinase 7 homolog of Xenopus MO15 cdk activating kinase CDK7 mRNA Cdk8 J6 _001260 Homo sapiens cyclin dependent kinase 8 CDK8 mRNA 1 CHK1 NM 001274 Homo sapiens CHK1 checkpoint S pombe homolog CHEK1 mRNA CHES1 K6 U68723 Human checkpoint suppressor 1 mRNA CNTF C11 X60542 Human gene for ciliary neurotrophic factor CNTFR D11 NM_001842 Homo sapiens ciliary neurotrophic factor receptor CNTFR mRNA CRADD RAIDD E19 NM_003805 Homo sapiens CASP2 and RIPK1 domain containing adaptor with death domain CRADD mRNA CREB1 18 NM_004379 Homo sapiens cAMP responsive element binding protein 1 CREB1 CREB2 J8 M86842 Human cAMP response element regulatory protein CREB2 gene CrmA J22 M14217 virus white pock variant CPV W2 crmA gene complete cds CRYAB F19 NM 001885 Homo sapiens crystallin alpha B CRYAB mRNA CSF 1 E11 M76453 Human colony stimulating factor 1 CSF1 gene CSNKI1A1 CK1 2 NM 001892 Homo sapiens casein kinase 1 alpha 1 CSNK1A1 mRNA Cyclin A1 L6 NM 003914 Homo sapiens cyclin A
38. zed H2O add 10 ml of 10X Detection Buffer stock solution Shake well before use Genemed Biotechnologies Inc 458 Carlton Court South San Francisco CA 94080 Tel 650 952 0110 Toll Free USA amp Canada 1 877 GENEMED 436 3633 Fax 650 952 1060 Email infoOgenemed com www genemed com Technical Support Tel 650 952 0110 Toll Free USA amp Canada 1 877 GENEMED 436 3633 Fax 650 952 1060 Email tech genemed com
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