Genemed Biotechnologies, Inc. PRODUCT …search.cosmobio.co.jp/cosmo_search_p/search_gate2/docs/...Genemed Biotechnologies, Inc. • 458 Carlton Court, South San Francisco, CA 94080

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: [email protected] • www.genemed.com

PRODUCT ANALYSIS CERTIFICATE

Star Profiler Array Nonradioactive System

FOR RESEARCH USE ONLY

CATALOG #: 05-100 SIZE: 1 KIT

Description: The Star ProfilerTM Array Nonradioactive System employs digoxigenin (DIG)-11-

dUTP-labeled cDNA and chemiluminescent detection. Each Star Profiler Array contains 373 hu-

man genes, which have been spotted onto a positively charged nylon membrane. The spots on the

array consists of popular and well-studied genes that are categorized in six functional groups:

oncogenes & tumor suppressors, cell-cycle, apoptosis, regulatory proteins, kinases, and cytokines &

receptors. Enough reagents are provided for four hybridizations.

Profiler Array and Hybridization Kit (Cat.# 02-100):2 Star Profiler Arrays

25 ml Hybridization Buffer

60 ml 20X SSC

60 ml 10% SDS

Profiler Array cDNA Synthesis Kit (Cat.# 02-200):10 µg Control K562 Total RNA

10 µl Reverse Transcriptase

15 µl Random Primer Mix

15 µl Probe Mix

50 µl 100mM DTT

100 µl 5X RT Buffer

100 µl dH20

Profiler Array Chemiluminescence Detection Kit (Cat.# 02-300):

15 ml CDP-Star Substrate

15 ml Alk-Phosphatase Conjugate

30 ml 5X Blocking Buffer

50 ml 10X Antibody Wash Buffer I

50 ml 10X Antibody Wash Buffer II

30 ml 10X Detection Buffer

Additional Materials1 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 year 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.

(PAC0075)

GenemedB i o t e c h n o l o g i e s , I n c .

cosmo



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Star Profiler Gene ListCONTROLS Coordinates Genbank#beta-actin P:2, 7, 9, 17, 21, 24 X00351, J00074, M10278

M24, N24, O24

Ubiquitin P1, P8, O9, P16 M26880

The following gene list is ordered alphabetically for your convenience.

Gene Coord. Genbank # Gene name14-3-3 O13 U28964 Human 14-3-3 protein

A-Raf-1 G2 X04790 Human mRNA for A-raf-1 (v-raf) oncogene

ABL-2 C3 NM_005158 Human v-abl Abelson murine leukemia viral oncogene

homolog 2 (arg, Abelson-related gene)

AKT1 D3 NM_005163 Human v-akt murine thymoma viral oncogene homolog 1

AKT2 E3 NM_001626 Human v-akt murine thymoma viral oncogene homolog 2

AKT3 F3 AF135794 Human AKT3 protein kinase

ANG-1 G24 U83508 Human angiopoietin-1 (ANG-1)

AP-2 a O10 NM_003220 Human transcription factor activating enhancer-binding

protein 2 alpha (TFAP2A)

Apaf-1 K11 AF013263 Human apoptotic protease activating factor 1 (Apaf-1)

APC G3 M74088 Human APC

API1 P10 NM_001166 Human apoptosis inhibitor 1 (API1)

ATF-B E14 NM_006399 Human activating transcription factor B (B-ATF)

ATF1 A14 NM_005171 Human activating transcription factor 1 (ATF1)

ATF2 B14 X15875 Human mRNA for cAMP response element (CRE-BP1)

binding protein

ATF3 C14 NM_001674 Human activating transcription factor 3 (ATF3)

ATF6 D14 AB015856 Human gene for ATF6

β-catenin H3 Z19054 Human beta catenin

β-myb D1 X13293 Human B myb oncogene

Bad A10 AF031523 Human bcl-xL/bcl-2 associated death promoter (BAD)

Bak B10 U16811 Human Bak

Bax-a C10 L22473 Human Bax alpha

Bcl-2 D10 M14745 Human bcl-2

Bcl-xL G10 Z23115 Human bcl-xL

Bcl2-b E10 M13995 Human B cell proto-oncogene

Bcl2-w F10 U59747 Human Bcl-w (bcl-w)

BCR I3 NM_004327 Human breakpoint cluster region (BCR)

BECN1 H6 AF139131 Human beclin 1 (BECN1)

BID H10 NM_001196 Human BH3 interacting domain death agonist (BID)

BIK I10 U34584 Human Bcl-2 interacting killer (BIK)

BimEL K10 AF032457 Human BimEL

BLK J20 Z33998 Human mRNA for human lymphoid tyrosine kinase

related to murine Blk

Bmi-1 J10 NM_005180 Human murine leukemia viral (bmi-1) oncogene homolog

BNIP-1 L10 NM_001205 Human BCL2/adenovirus E1B 19kD-interacting protein 1

BNIP-2 M10 NM_004330 Human BCL2/adenovirus E1B 19kD-interacting protein 2

BNIP-3 N10 NM_004052 Human BCL2/adenovirus E1B 19kD-interacting protein 3

BRCA1 K3 NM_000058 Human breast cancer 1

BRCA2 L3 U43746 Human breast cancer 2

Brk K20 X78549 Human brk gene for tyrosine kinase

BRX J3 AF126008 Human breast cancer nuclear receptor-binding auxiliary

protein (BRX)

Bst1 M3 NM_004334 Human bone marrow stromal cell antigen 1 (BST1)

Btk L20 NM_000061 Human Bruton agammaglobulinemia tyrosine kinase(BTK)

Bub1 E9 AF053305 Human mitotic checkpoint kinase Bub1 (BUB1)

Bub3 F9 AF081496 Human kinetochore protein BUB3 (BUB3)

c-abl (ABL-1 ) B3 NM_005157 Human v-abl Abelson murine leukemia viral oncogene

homolog 1



c-cbl (CBL) N3 X57110 Human c-cbl proto-oncogene

c-crk O3 NM_005206 Human v-crk avian sarcoma virus CT10 onco homolog (CRK)

c-erbB2 (ErbB2) C4 X03363 Human c-erb-B-2

c-fes E4 X52192 Human c-fes/fps proto-oncogene

c-fgr (scr) K2 M19722 Human Proto-oncogene

c-fms D4 X03663 Human c-fms proto-oncogene

c-fos F1 K00650 Human Proto-oncogene

c-fyn (scr) L2 NM_002037 Human FYN oncogene related to SRC, FGR, YES (FYN)

c-jun H1 J04111 Human c-jun proto oncogene

c-kit J4 X06182 Human Proto-oncogene

c-Lck K4 NM_005356 Human lymphocyte-specific protein tyrosine kinase (LCK)

c-lyn (scr) M2 M16038 Human lyn mRNA encoding a tyrosine kinase

c-maf L4 NM_005360 Human v-maf musculoaponeurotic fibrosarcoma (avian)

oncogene

c-mas M4 M13150 Human mas proto-oncogene

c-met N4 M60718 Human hepatocyte growth factor

c-mos O4 J00119 Human humos gene homologous to transforming gene ofmmsv

c-mpl N21 M90102 Human (clones 15, 39, 41) c-myeloproliferative leukemia virus

type P (c-mpl-P)

c-myb E1 M15024 Human Proto-oncogene

c-myc A1 E01841 Human proto-oncogene

c-Pim2 B5 NM_006875 Human proto-oncogene Pim-2 (PIM2) serine threonine kinase

c-raf-1 H2 X03484 Human mRNA for raf oncogene

c-ral A A3 NM_005402 Human v-ral simian leukemia viral oncogene homolog A (ras

related) (RALA)

c-rel C5 NM_002908 Human v-rel avian reticuloendotheliosis viral oncogene

homolog (REL)

c-ros1 J5 NM_002944 Human v-ros avian UR2 sarcoma virus oncogene homolog 1

c-ski G5 X15218 Human ski onco

c-src (scr) J2 X59932 Human C-SRC-kinase

c-syn (scr) N2 M14333 Human c-syn protooncogene

c-yes O2 M15990 Human c-yes-1

C3G B13 D21239 Human C3G

CAD G12 AB013918 Human mRNA for CAD

Calcineurin A1 B17 M29550 Human calcineurin A1

Calcineurin B C17 M30773 Human calcineurin B

Caspase-1 A11 NM_001223 Human apoptosis-related cysteine protease

Caspase-10 B11 NM_001230 Human apoptosis-related cysteine protease

Caspase-2 C11 NM_001224 Human apoptosis-related cysteine protease

Caspase-3 D11 NM_004346 Human apoptosis-related cysteine protease

Caspase-4 E11 NM_001225 Human apoptosis-related cysteine protease

Caspase-5 F11 NM_004347 Human apoptosis-related cysteine protease

Caspase-6 G11 NM_001226 Human apoptosis-related cysteine protease

Caspase-7 H11 NM_001227 Human apoptosis-related cysteine protease

Caspase-8 I11 NM_001228 Human apoptosis-related cysteine protease

Caspase-9 J11 NM_001229 Human apoptosis-related cysteine protease

Cdc42 D2 NM_001791 Human cell division cycle 42 (GTP-binding protein, 25kD)

Cdc45 J16 AF062495 Human Cdc45

Cdk1 (Cdc2) A7 X05360 Human CDC2 gene involved in cell cycle control

Cdk2 B7 NM_001798 Human cyclin-dependent kinase 2 (CDK2)

Cdk3 C7 NM_001258 Human cyclin-dependent kinase 3 (CDK3)

Cdk4 D7 NM_000075 Human cyclin-dependent kinase 4 (CDK4)

Cdk5 E7 NM_004935 Human cyclin-dependent kinase 5 (CDK5)

Cdk6 F7 NM_001259 Human cyclin-dependent kinase 6 (CDK6)

Cdk7 G7 NM_001799 Human cyclin-dependent kinase 7 (CDK7), homolog of

Xenopus MO15 cdk-activating kinase

Cdk8 H7 NM_001260 Human cyclin-dependent kinase 8 (CDK8)

Gene Coord. Genbank # Gene name



Cdk9 D9 NM_001261 Human cyclin-dependent kinase 9 (CDC2-related kinase; CDK9)

CHES1 N9 U68723 Human checkpoint suppressor 1

CIP2 A17 L25876 Human protein tyrosine phosphatase (CIP2)

Cks1 G9 NM_001826 Human CDC28 protein kinase 1 (CKS1)

CLK H9 NM_004071 Human CDC-like kinase (CLK)

c-cot P3 D14497 Human mRNA for proto-oncogene protein

CPAN A12 AF039210 Human caspase-activated nuclease

CREB1 F14 NM_004379 Human cAMP responsive element binding protein 1 (CREB1)

CREB2 G14 M86842 Human cAMP response element regulatory protein (CREB2)

CSF1 P22 M76453 Human colony stimulating factor 1 (CSF1)

Cyclin A1 A8 NM_003914 Human cyclin A1 (CCNA1)

Cyclin A2 B8 X51688 Human cyclin A

Cyclin B1 C8 M25753 Human cyclin B mRNA, 3' end

Cyclin B2 D8 AB020981 Human cyclin B2

Cyclin C E8 NM_005190 Human cyclin C (CCNC)

Cyclin D1 F8 M64349 Human cyclin D1

Cyclin D2 G8 NM_001759 Human cyclin D2 (CCND2)

Cyclin D3 H8 M90814 Human D3-type cyclin (CCND3)

Cyclin E I8 M73812 Human cyclin E mRNA sequence

Cyclin E2 J8 AF102778 Human cyclin E2

Cyclin F K8 NM_001761 Human cyclin F (CCNF)

Cyclin G1 L8 U53328 Human cyclin G

Cyclin G2 M8 NM_004354 Human cyclin G2 (CCNG2)

Cyclin H N8 U12685 Human cyclin H

Cyclin I O8 NM_006835 Human cyclin I (CCNI)

Cyclin K A9 AF060515 Human cyclin K (CPR4)

Cyto C1 M11 NM_001916 Human cytochrome c-1 (CYC1)

DAPK P12 X76104 Human DAP-kinase

Dbl A4 X12556 Human dbl proto-oncogene

DCC B4 X76132 Human DCC

DDX1 B12 NM_004939 Human DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 1

DFF45 C12 U91985 Human DNA fragmentation factor-45

DHFR I16 NM_000791 Human dihydrofolate reductase (DHFR)

E1A K6 M18288 Adenovirus type 40 E1A and E1B

E1B19k L6 M18289 Adenovirus type 40 E1A and E1B

E1B55K M6 M18289 Adenovirus type 40 E1A and E1B

E2F-1 H14 M96577 Human (E2F-1) pRB-binding protein

E2F-2 I14 NM_004091 Human E2F transcription factor 2 (E2F2)

E2F-3 J14 Y10479 Human mRNA for E2F-3 transcription factor

E2F-4 K14 NM_001950 Human E2F transcription factor 4, p107/p130-binding (E2F4)

E2F-5 L14 NM_001951 Human E2F transcription factor 5, p130-binding (E2F5)

E2F-6 M14 AF059292 Human transcriptional repressor E2F-6

EGF A21 NM_001963 Human epidermal growth factor (EGF)

EGFR B21 NM_005228 Human epidermal growth factor receptor (avian erythroblas

tic leukemia viral [v-erb-b] oncogene homolog

EGR1 B16 NM_001964 Human early growth response 1 (EGR1)

EIF3S6 E16 NM_001568 Human Int-6

EIF4B D16 NM_001417 Human eukaryotic translation initiation factor 4B (EIF4B)

ELK C19 M25269 Human tyrosine kinase (ELK1) oncogene

EPO K21 NM_000799 Human erythropoietin (EPO)

EPOR L21 NM_000121 Human erythropoietin receptor (EPOR)

ER L24 X03635 Human mRNA for oestrogen receptor

ERK1 A18 X60188 Human ERK1 mRNA for protein serine/threonine kinase

ERK3 B18 X80692 Human ERK3

ERK5 C18 U25278 Human ERK5

Ets-1 F4 J04101 Human erythroblastosis virus oncogene homolog 1 (ets-1)

Ets-2 G4 NM_005239 Human v-ets avian erythroblastosis virus E26 oncogene




homolog 2 (ETS2)

FADD D12 NM_003824 Human Fas (TNFRSF6)-associated via death domain (FADD)

FAK M20 L13616 Human focal adhesion kinase (FAK)

Fas O11 M67454 Human Fas Antigen

Fas-ligand P11 D38122 Human Fas ligand

FGF-1 C21 E03692 cDNA encoding human acidic FGF

FGF-2 D21 M27968 Human basic fibroblast growth factor (FGF)

FGF-3 E21 NM_005247 Hu. fibroblast growth factor 3 (murine mammary tumor virus

integration site (v-int-2) oncogene homolog)

FGF-4 F21 NM_002008 Human fibroblast growth factor 4 (heparin secretory

transforming protein 1, Kaposi sarcoma oncogene)

FGFr G21 M34185 Human fibroblast growth factor receptor (FGFr) transmem

brane form

FHIT G6 NM_002012 Human fragile histidine triad gene (FHIT)

FKBP1A G17 NM_000801 Human FK506-binding protein 1A (12kD; FKBP1A)

FLI-1 K5 X67001 Human HUMFLI-1

FLT1 F24 AF063657 Human vascular endothelial growth factor receptor (FLT1)

fos B G1 NM_006732 Human FBJ murine osteosarcoma viral oncogene homolog B

FRAP H17 L34075 Human FKBP-rapamycin associated protein (FRAP)

FRP-1 I17 U49844 Human FRAP-related protein (FRP1)

G3BP2 A13 AF051311 Human Ras-GAP SH3 binding protein (G3BP2)

GADD-45 L9 M60974 Human growth arrest and DNA-damage-inducible protein

GAK I9 NM_005255 Human cyclin G associated kinase (GAK)

GCSF O21 NM_000759 Human colony stimulating factor 3 (granulocyte) (CSF3)

Gli H4 NM_005269 Human glioma-associated oncogene homolog (zinc finger protein)

Gli2 I4 NM_005270 Human GLI-Kruppel family member GLI2

GMCSF A22 NM_000758 Human colony stimulating factor 2 (granulocyte-macrophage)

Granzyme B N11 M28879 Human granzyme B (CTLA-1)

GRF2 C13 NM_005312 Human guanine nucleotide-releasing factor 2 (specific for crk

proto-oncogene) (GRF2)

GRP1 D13 AJ005197 Human mRNA for guanine nucleotide exchange factor GRP1

Ha-ras A2 M30708 Synthetic human c-Ha-ras gene encoding p21 protein

HIF-1a P13 U22431 Human hypoxia-inducible factor 1 alpha (HIF-1 alpha)

HPV E6 N6 K02718 Human papillomavirus type 16 (HPV16), complete genome

HPV E7 O6 K02718 Human papillomavirus type 16 (HPV16), complete genome

HSP27 F16 U15590 Human heat shock 17kD protein 3 (HSPB3)

Hsp70-1 G16 NM_005346 Human heat shock 70kD protein 1 (HSPA1B)

Hsp90 H16 X15183 Human 90-kDa heat-shock protein

IFN-a J22 A15692 Human IFN-alpha 1

IFN-b K22 M35591 Human fibroblast interferon (IFN-beta)

IFN-g L22 J00219 Human immune interferon (IFN-gamma)

IGF-1 M22 M29644 Human insulin-like growth factor I

IGF-2 N22 M29645 Human insulin-like growth factor II

IkB (MAD3) I13 M69043 Human MAD-3 mRNA encoding IkB-like activity

IKK-a D19 AF080157 Human IkB kinase-alpha (IKK-alpha)

IKK-b E19 AF080158 Human IkB kinase-beta (IKK-beta)

IKK-g F19 AF074382 Human IkB kinase gamma subunit (IKK-gamma)

IL-10 O23 U16720 Human interleukin 10 (IL10)

IL-10R P23 U00672 Human interleukin-10 receptor (IL-10R)

IL-11 A24 M57765 Human interleukin 11

IL-12 (p35) B24 AF180562 Human interleukin 12, p35

IL-12 (p40) C24 AF180563 Human interleukin 12, p40

IL-1a A23 X03833 Human gene for interleukin 1 alpha (IL-1 alpha)

IL-1R2 B23 NM_004633 Human interleukin 1 receptor, type II (IL1R2)

IL-2 C23 U25676 Human interleukin 2

IL-2R D23 K03122 Human interleukin-2 receptor mRNA (short form)

IL-3 E23 M14743 Human interleukin 3 (IL-3)




IL-3Ra F23 M74782 Human interleukin 3 receptor (hIL-3Ra)

IL-4 G23 NM_000589 Human interleukin 4 (IL4)

IL-5 H23 J03478 Human interleukin 5 (IL-5)

IL-6 I23 M54894 Human interleukin 6

IL-6R J23 X12830 Human mRNA for interleukin-6 (IL-6) receptor

IL-7 K23 J04156 Human interleukin 7 (IL-7)

IL-7R L23 M29696 Human interleukin-7 receptor (IL-7)

IL-8 M23 M28130 Human interleukin 8 (IL8)

IL-8R N23 M68932 Human IL-8 receptor (IL-8R)

ING1 (p33) I6 AF110645 Human candidate tumor suppressor p33 ING1 homolog

Insulin D24 J00265 Human insulin

IRF-1 N16 NM_002198 Human interferon regulatory factor 1 (IRF1)

IRF-4 O16 U52682 Human lymphocyte specific interferon regulatory factor/

interferon regulatory factor 4 (LSIRF/IRF4)

JAK1 G19 M64174 Human protein-tyrosine kinase (JAK1)

JAK2 H19 NM_004972 Human Janus kinase 2 (a protein tyrosine kinase) (JAK2)

JAK3 I19 U09607 Human JAK family protein tyrosine kinase (JAK3)

JNK1 A19 NM_002750 Human mitogen-activated protein kinase 8 (MAPK8)

Jun B I1 X51345 Human jun-B mRNA for JUN-B protein

Jun D J1 X51346 Human jun-D mRNA for JUN-D protein

Ki-ras B2 L00049 Human cellular c-Ki-ras2 proto-oncogene

Krs-1 G20 U60206 Human stress responsive serine/threonine protein kinase Krs-1

L-myc B1 M19720 Human L-myc protein

Large-TAntigen P6 J02400 Simian virus 40 Large T Antigen

Mad J13 L06895 Human antagonizer of myc transcriptional activity

MADD E12 NM_003682 Human MAP-kinase activating death domain (MADD) mRNA

max K13 X60287 Human max gene

MDM2 P4 Z12020 Human p53-associated gene

MEF2A F13 NM_005587 Human MADS box transcription enhancer factor 2,

polypeptide A (myocyte enhancer factor 2A; MEF2A)

MEK1 D18 L11284 Human ERK activator kinase (MEK1)

MEK2 E18 L11285 Homosapiens ERK activator kinase (MEK2)

MEK3 F18 NM_002756 Human mitogen-activated protein kinase kinase 3 (MAP2K3)

MEK5 H18 NM_002757 Human mitogen-activated protein kinase kinase 5 (MAP2K5)

MEK6 I18 U39657 Human MAP kinase kinase 6 (MKK6)

MEKK1 L18 AAC97073 MEK kinase 1 (see also AF042838)

MEKK2 M18 NM_006609 Human mitogen-activated protein kinase kinase kinase 2(MAP3K2)

MEKK3 N18 U78876 Human MEK kinase 3

MEKK4 O18 NM_005922 Human mitogen-activated protein kinase kinase kinase 4

(MAP3K4) transcript variant 1

MEKK5 (ASK1) P18 NM_005923 Human mitogen-activated protein kinase kinase kinase 5(MAP3K5)

MEN1 F6 U93236 Human menin (MEN1)

MKK4 G18 U17743 Human JNK activating kinase (JNKK1)

MKK7 J18 AF014401 Human JNK kinase 2 (JNKK2)

MLK3 K18 L32976 Human protein kinase (MLK-3)

MMP1 J24 U41078 Human membrane-type matrix metalloproteinase-1

MSK1 F20 AF074393 Human nuclear mitogen- and stress-activated protein kinase-1

MTCP-1 M5 Z24459 Human MTCP1 gene, exons 2A to 7 (and joined mRNA)

MTF1 P15 NM_005955 Human metal-regulatory transcription factor 1 (MTF1)

MyoD L13 X56677 Human MyoD

n-myc C1 Y00664 Human germ line n-myc

N-ras C2 M14307 Synthetic N-ras

NCK1 H13 NM_006153 Human NCK adaptor protein 1 (NCK1)

NFAT-1B N14 U43341 Human transcription factor NFAT1 isoform B (NFAT1)

NFAT4 O14 L41067 Human NF-AT4c

NFAT5 P14 AF134870 Human transcription factor NFAT5

NFkB2 O17 X61498 Human mRNA for NF-kB subunit




NGF-2 B22 X53655 Human mRNA for NGF-2

NGF-B C22 NM_002506 Human nerve growth factor, beta polypeptide (NGFB)

NGFR D22 M14764 Human nerve growth factor receptor

NIK J19 Y10256 Human mRNA for serine/threonine protein kinase

NOS C16 AF068236 Human inducible nitric oxide synthase (NOS)

NTRK1 H20 X66397 Human tpr mRNA

ODC A16 M34158 Human ornithine decarboxylase (ODC)

p107 N1 L14812 Human retinoblastoma related protein (p107)

p110 (Rb) M1 M28419 Human retinoblastoma susceptibility protein

p125 (XP-C) N17 D21089 Human mRNA for XP-C repair complementing protein (p125)

p130 (Rb2) O1 X74594 Human Rb2/p130 protein

p15 I7 L36844 Human (clone p15INK4B/HA5) CDK inhibitory protein

p16 J7 L27211 Human CDK4-inhibitor (p16-INK4)

p18 K7 U17074 Human CDK6 inhibitor p18

p19 L7 U40343 Human CDK inhibitor p19INK4d

p21 M7 L25610 Human cyclin-dependent kinase inhibitor

p27 N7 X84849 Human p27kip1

p300 M13 U01877 Human p300 protein

p38 B19 L35253 Human p38 mitogen activated protein (MAP) kinase

p53 K1 K03199 Human p53 Tumor Antigen

p57 O7 U48869 Human cdk-inhibitor p57/KIP2 (CDKN1C)

p58 (XP-C) M17 D21090 Human mRNA for XP-C repair complementing protein (p58/

HHR23B)

p70 (S6K) C20 AB019245 Human p70 S6Kb S6 kinase b

p73 L1 Y11416 Human mRNA for P73

p90 (rsk) D20 L07597 Human ribosomal protein S6 kinase 1 (RPS6KA1)

p97 C6 M12154 Human melanoma-associated antigen p97 (melanotransferrin)

PA2G4 M9 NM_006191 Human proliferation-associated 2G4, 38kD (PA2G4)

PAK1 E20 NM_002576 Human p21/Cdc42/Rac1-activated kinase 1 (yeast Ste20-related)

PARP L11 NM_001618 Human ADP-ribosyltransferase (NAD+; poly [ADP-ribose]

polymerase; ADPRT)

PCNA K9 NM_002592 Human proliferating cell nuclear antigen (PCNA)

PDGF R J21 J03278 Human platelet-derived growth factor (PDGF) receptor

PDGF-A H21 X06374 Human mRNA for platelet-derived growth factor PDGF-A

PDGF-B (c-sis) I21 X02811 Human platelet-derived growth factor B chain (PDGF-B)

PI3K K19 Y11312 Human gene for phosphoinositide 3-kinase

Pim-1 A5 NM_002648 Human pim-1 oncogene (PIM1)

PISSLRE C9 NM_003674 Human cyclin-dependent kinase (CDC2-like) 10 (CDK10)

PITSLARE A1 B9 U04815 Human protein kinase PITSLRE alpha 1

PKA-a L19 X07767 Human mRNA for cAMP-dependent protein kinase catalytic

subunit type alpha

PKC-a M19 X52479 Human PKC alpha protein kinase C alpha

PKC-b1 N19 X06318 Human gene protein kinase C (PKC) type beta I

PKC-d P19 NM_006254 Human protein kinase C, delta (PRKCD)

PKC-g O19 Z15114 Human gene for protein kinase C gamma

PKC-q A20 NM_006257 Human protein kinase C, theta (PRKCQ)

PKC-z B20 Z15108 Human protein kinase C zeta

PLC-g G13 M34667 Human phospholipase C-gamma

PML J17 NM_002675 Human promyelocytic leukemia (PML)

PMS1 K17 NM_000534 Human postmeiotic segregation increased (S. cerevisiae) 1

PMS2 L17 NM_000535 Human postmeiotic segregation increased (S. cerevisiae) 2

PSA B6 NM_001648 Human antigen prostate specific (APS)

PTEN E6 U93051 Human putative protein tyrosine phosphatase (PTEN)

RAC1 E2 NM_006908 Hu. ras-related C3 botulinum toxin substrate 1 (rho family,

small GTP binding protein Rac1)

Raf-B I2 NM_004333 Human v-raf murine sarcoma viral oncogene homolog B1

Ref1 D17 S43127 ref-1=redox factor




RelA D5 L19067 Human NF-kappa-B transcription factor p65 subunit

RelB E5 NM_006509 Hu. v-rel avian reticuloendotheliosis viral oncogene homolog B

(NFk light polypeptide enhancer in B-cells3)

Ret F5 X12949 Human ret proto-oncogene for tyrosine kinase

RhoA F2 L25080 Human GTP-binding protein (rhoA)

RIP-1 E17 NM_007043 Human Rev interacting protein (RIP-1)

RIPK1 H12 NM_003804 Human receptor (TNFRSF)-interacting serine-threonine kinase 1

SCF O22 M59964 Human stem cell factor

SFRS1 K16 NM_006924 Human splicing factor, arginine/serine-rich 1 (splicing factor 2,

alternate splicing factor; SFRS1)

SFRS2 L16 NM_003016 Human splicing factor, arginine/serine-rich 2 (SFRS2)

SHC I20 U73377 Human p66shc (SHC)

Sky N20 D17517 Human sky mRNA for Sky

Smad1 A15 U54826 Human mad-related protein MADR1

Smad2 B15 U65019 Human MAD-related protein 2 (MADR2)

Smad4 C15 U44378 Human homozygous deletion target in pancreatic carcinoma

(DPC4)

Smad5 D15 U73825 Human Smad5

Smad6 E15 AF035528 Human Smad6

Smad7 F15 AF015261 Human Smad7 protein

SOS E13 NM_005633 Human son of sevenless (Drosophila) homolog 1 (SOS1)

SRF N13 NM_003131 Human serum response factor (c-fos serum response element-

binding transcription factor; SRF)

Stat1 G15 M97935 Human transcription factor ISGF-3

Stat2 H15 NM_005419 Human signal transducer and activator of transcription

2, 113kD (STAT2)

Stat3 I15 AJ012463 Human mRNA for transcription factor

Stat4 J15 L78440 Human STAT4

Stat5A K15 U43185 Human signal transducer and activator of transcription

Stat6 L15 NM_003153 Human signal transducer and activator of transcription

6,interleukin-4 induced (STAT6)

Syk O20 NM_003177 Human spleen tyrosine kinase (SYK)

TCF-1A M15 X59869 Human TCF-1 mRNA for T cell factor 1 (splice form A)

TCF-4 N15 Y11306 Human mRNA for hTCF-4

TCF19 O15 NM_007109 Human transcription factor 19 (SC1) (TCF19)

TCL-1 N5 X82240 Human mRNA for Tcell leukemia/lymphoma 1

TCL-3 O5 M62626 Human T lymphocyte homeobox protein (partial cds)

TDP-43 F17 U23731 Human TAR DNA-binding protein-43

TEK P20 NM_000459 Human TEK tyrosine kinase, endothelial (TEK)

TGF-a E22 M31172 Human transforming growth factor-alpha

TGF-b F22 X02812 Human transforming growth factor-beta

TGFb-RI G22 NM_004612 Human transforming growth factor, beta receptor I (activin A

receptor type II-like kinase, 53kD; TGFBR1)

TGFb-RII H22 NM_003242 Human transforming growth factor, beta receptor II (70-80

kD) (TGFBR2)

TGFb-RIII I22 NM_003243 Human transforming growth factor, beta receptor III

(betaglycan, 300 kD) (TGFBR3)

THRA L5 M24748 Human thyroid hormone receptor alpha 1 (TR-alpha-1)

TIE-1 H24 AF023676 Human lamin B receptor homolog TM7SF2 (TM7SF2)

TIM H5 NM_005435 Human oncogene TIM (TIM)

TIMP-1 I24 NM_003254 Human tissue inhibitor of metalloproteinase 1 (erythroid

potentiating activity, collagenase inhibitor) TIMP1

TNF L12 X01394 Human tumor necrosis factor

TNF-R1 M12 X55313 Human TNF-R gene for tumor necrosis factor receptor type 1

TNF-R2 N12 U52156 Human tumor necrosis factor receptor 2 (TNFR2)

TPA K24 M18182 Human plasminogen activator

TPO M21 NM_000460 Hu. thrombopoietin(myeloproliferative leukemia virus




oncogene ligand, megakaryocyte growth&development factor)

TRADD F12 L41690 Human TNF receptor-1 associated protein (TRADD)

TRAF1 I12 NM_005658 Human TNF receptor-associated factor 1 (TRAF1)

TRAF4 J12 NM_004295 Human TNF receptor-associated factor 4 (TRAF4)

TRAF6 K12 NM_004620 Human TNF receptor-associated factor 6 (TRAF6)

TRAIL O12 U37518 Human TNF-related apoptosis inducing ligand TRAIL gene

TUNP D6 X72727 Human tunp mRNA for transformation upregulated nuclear

protein

U2AF M16 X64044 Human mmRNA for large subunit of splicing factor U2AF

v-src J6 D10652 Rous sarcoma virus genome

vav P5 X16316 Human vav oncogene

VEGF E24 E14233 Human mRNA for vascular endothelial growth factor (VEGF)

WEE1 J9 NM_003390 Human wee1+ (S. pombe) homolog (WEE1)

wnt-1 A6 NM_005430 Human wingless-type MMTV integration site family, member 1

WT1 I5 X51630 Human Wilms tumor WT1 mRNA for zinc finger protein,

Krueppel-like




TrademarkProfilerTM is a trademark of Genemed Biotechnologies, Inc.

CDP-Star® is a registered trademark of Tropix, Inc.

WARRANTYGENEMED 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.

ProfilerTM

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)

GenemedB i o t e c h n o l o g i e s , I n c .

Page 2 Genemed Biotechnologies, Inc.

Profiler Array Nonradioactive System

TABLE OF CONTENTS

I. Introduction 3

II. Package Contents 5

III. Additional Materials & Equipment 6

IV. Instructions for Use 6

V. Troubleshooting Guide 10

VI. Appendix 11

TRADEMARKSProfilerTM is a trademark of Genemed Biotechnologies, Inc.

CDP-Star® is a registered trademark of Tropix, Inc.

GenBank® is a registered trademark of The United States Department of Health and Human Services.

FluorChemTM is a trademark of Alpha Innotech Corporation.

HyperfilmTM is a trademark of Amersham Pharmacia Biotech.

Saran Wrap® is a registered trademark of S.C. Johnson.

WARRANTYGENEMED 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.

Genemed Biotechnologies, Inc. Page 3


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 ProfilerTM 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 (>90 nt), synthesized

from a carefully selected sequence. Our selection process eliminates any ho-

mologous 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 ProfilerTM ArrayUnlike 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.



Figure 1 provides a simplified overview of the Profiler procedure. After cDNA

labeling, Profiler Arrays are hybridized overnight with DIG-11-dUTP-labeled

cDNA. The next day, the 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

x-ray film or by using a chemiluminescent-imaging system, such as Alpha

Innotech's FluorChem Imaging System.

I. INTRODUCTION continued

Total Time:

1–2 days (~24–28 hr)

Isolate Total RNA

(~1–6 hr)

Prepare Labeled cDNA &

Prehybridize Profiler Arrays

(~2–3 hr)

Hybridize overnight

(~12 hr)

Detect & analyze

(~3–4 hr)

Sample 1(e.g., control)

TTTTAAAA AAAA

Hybridization Bag

array 1 array 2

Obtain cell or tissue samples

(5-min exposure to x-ray film)

Sample 2(e.g., experimental)

AAAA

AAAA

AAAA

AAAA

DIG DIGTTTT

DIG DIG



II. PACKAGE CONTENTS

See the front cover of this User Manual for appropriate storage conditions.

For Buffer Solutions & 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

• 25 ml Hybridization Buffer

• 60 ml 20X SSC

• 60 ml 10% SDS

Profiler Array cDNA Synthesis Kit (Store at –20°C)

• 10 µg Control K562 Total RNA (yellow)

• 10 µl Reverse Transcriptase (amber)

• 15 µl Random Primer Mix (blue)

• 15 µl Probe Mix (pink)

• 50 µl 100mM DTT (salmon)

• 100 µl 5X RT Buffer (purple)

• 100 µl dH20 (natural)

Profiler Array Chemiluminescence Detection Kit (Store at 4°C)

• 15 ml CDP-Star Substrate

• 30 ml 5X Blocking Buffer

• 50 ml 10X Antibody Wash Buffer I

• 50 ml 10X Antibody Wash Buffer II

• 30 ml 10X Detection Buffer

• 15 µl Alk-Phosphatase Conjugate (orange)

Additional Materials Provided

• 1 Profiler Grid

• 1 Product User Manual

†See Product Analysis Certificate provided with each kit for complete list of genes spotted on the array.



III. ADDITIONAL MATERIALS & 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• 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

IV. INSTRUCTIONS FOR USE

A. Isolation of total RNA from cells or tissuesTotal 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 A260

/A280

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 µg of RNA on a denaturing formaldehyde/agar-

ose/ethidium-bromide gel. Two bright bands, 28S and 18S rRNA, should be

observed at approximately 5 and 2 kb, respectively. These bands are sharply

defined and visible at about a 2:1 ratio (28S:18S).

B. Preparation of cDNA probeFor 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°C.



2. For each total RNA sample, combine the following components into a

sterile 0.5-ml microcentrifuge tube:

5–10 µg Total RNA

3 µl Random Primer Mix

Bring each tube to a final volume of 10 µl by adding dH2O (provided). Mix

well by pipetting and spin briefly in a microcentrifuge. Keep on ice.

3. 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.

4. Prepare the cDNA synthesis mixture for each total RNA sample:

6 µl 5X First Strand Buffer†

3 µl 0.1 M DTT†

6 µl Probe Mix

1 µl Reverse Transcriptase

4 µl H2O (provided)

20 µl Total volume per RNA sample

5. Mix well by pipetting. Incubate the cDNA synthesis mixture at 42°C for 2 min.

6. Transfer the mixture to the tube containing the RNA sample (from Step

B3) and mix well by pipetting. Then incubate at 42°C for 2 hr.

7. 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°C until use.

C. Pre-HybridizationBefore you start, preheat Hybridization Buffer to 52°C. Completely dissolve all

precipitates in the Hybridization Buffer. Prepare sheared salmon testes DNA:

1. Heat the sheared salmon testes DNA (~10 mg/ml) at 95–100°C for 5 min,

then immediately place on ice (~5 min). We recommend preparing aliquots

of 100 µl (i.e., 50 µl per membrane), then storing at –20°C until use.

2. 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 H2O; avoid adding

H2O directly onto the membrane. Then carefully decant water.

4. For each membrane, prepare pre-hybridization solution: Add 50 µl 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 ≥2 hr.

IV. INSTRUCTION FOR USE continued



IV. INSTRUCTIONS FOR USE continued

D. Hybridization1. Heat DIG-labeled cDNA probe at 94°C for 5 min. Then incubate on ice

for 5 min.

2. Cut open 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 µl pipet tip container.

Quicky rinse 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.

E. DetectionNote 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 µl 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

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.



IV. 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 (i.e., 2–10 min). Alternatively, use a chemi-

luminescent-imaging system, such as Alpha Innotech's FluorChem Imager

or equivalent.

F. Interpretation of resultsFor quantitative results, we suggest using an chemiluminescent-imaging system

such as Alpha Innotech's FluorChem or equivalent. Be sure to read Section G

(below) to find out how to normalize the genes on the arrays. For a quick,

cursory 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.

G. NormalizationOn 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.



The following list of tips is designed to help you troubleshoot problems that you may

encounter when performing the procedure. If you feel you need additional technical

assistance, please contact us by phone, fax, or email.

A. Problem: high background

•Was the 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.

•Is the film overexposed?Try several exposures for varying lengths of time. This is necessary for ob-

taining optimal results.

•How much antibody did you add to the Blocking Buffer (in Step E3)?Make sure the antibody is diluted 1:5,000.

B. Problem: hybridization signals weak or absent

• Is the problem with your RNA quality?Be sure to check the quality of your RNA (Section IV.A).

•Are the kit reagents working properly?Strip the membrane and use the control K562 RNA (provided) to confirm

that reagents and materials are working properly.

•Did you hybridize the membrane at the correct temperature?Check to make sure the hybridization temperature is set at 52°C.

•How long was the 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).

•How long was the exposure?Try several exposures for varying lengths of time. This is necessary for ob-

taining optimal results.

V. TROUBLESHOOTING GUIDE



VI. APPENDIX

TO

P

BO

TTO

M

Notc

h

Beta

-acti

n s

pots

Array Orientation

VI. APPENDIX continued

Solution/Dilution Recipes (enough for two array membranes)

250 ml of 2X SSC/0.5% SDSTo 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% SDSTo 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 BufferTo 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 ITo 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 IITo 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 BufferTo 90 ml of deionized H2O, add 10 ml of 10X Detection Buffer stock solution.

Shake well before use.

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