Protein/DNA Arrays
Contents
1. 5 What this Manual Covers sch ane GA aa 040 ren 5 Safety Warnings and Precautions 5 For More 5 About the TranSignal Protein DNA Assay Kit 6 TranSignal Protein DNA Assay 6 EL 6 TranSignal Protein DNA Assay Kit Contents and Handling Conditions 6 Kit Contents and 5 6 Iit Handling s eru sake ceed eae fn esee ae Des 6 Required Materials and Equipment Not Provided 7 EQUIPMENT x VT a VS R eene prier esee 7 Sample Type Specific 7 Intr duction se xx elg nente hee RE E ME E n 7 Princicple of the Technology 7 Overview of Assay 8 rcc E 8 Guidelines for Assay Design and Analysis 9 Preparing 9 General Guidelines 9 Assay Procedures sexus Ep p VS vad XO Vee ea ak eier 9 Before You Start darasa O nennen nenn 9 Preparing Nuclear Extracts2. 9 Preparing the 9 Probe Mikes makane e e etd e t ce ata Dh er 10 ISOLATING TF BOUND PROBES 10 HYBRIDIZATION Ed AL 2 ern aa LG 11 DETECTION Be aes 11 RESULTS amp ANALYSIS c a Dola dts if h cun dae dr ee ee 12 REFERENCES 22 tere eru uetus EH oue etium ues a 12 APPENDIX Ede EN cH ee ERR Ed UE named 13 RECIPES AND 0 13 TranSignal Protein DNA Arrays User Manual iii About the User Manual About the User Manual Who Should Read this Manual What this Manual Covers Safety Warnings and Precautions For More Information Anyone that has purchased a Protein DNA Array Kit from Panomics to profile from 56 to 345 different Transcription Factors This manual provides recommendations and step by step procedures for the following Sample and assay preparation Assay procedure Troubleshooting Layouts CAUTION All chemicals should be considered potentially hazardous We recommend that this product and its components be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice For information about the Protein DNA products mentioned in this manual visit our website at www panomics com Protein DNA Arrays User Manual Page 5 About the Protein DNA Assay Kit About the Protein DN
3. am appudosddo asaym ainjoiajt aj ui paytsjqnd ueaq 4 awns 104 sexuenbes snsuasuo edijjnui siopn4 uondipsupij awos 104 050 10 s ojuo9 aatysod sp pasn aio pup pein uiotq payods suunjo smoi A Any 9I Gl EL CL LL OL 6 8 L 9 G v L ra AX ra 149 TT T 0 2 1 1 4 dd daaym dam j W waa a gx rau pun quus usegus day c Z day dd lA gur Teal nl Hel 99405 96 05 1 1 7 7 g IDU 4 ass S s qus ais us LIEIS Das IPS ous gravs grays 198 11 L98W 11 m y n id f ynd End bid Lu _ _ _ 2 Z 1 1 diad Gnd LION 18 1048 2481044 248109 808 soa a ay pup pay 9 d 9 ud Z Z 4 4 c 2 c Z Jus Odd cas Taga DE Dd Pa 9X6 cyw LXW DX Z z Z Z 4 4 D UW cp ga 0 20 po po DO suy suy 0 2 1 4 Ld c Z 7 _ 2 c _ _ PUN 93N 930 zan ZA an AVN dN GAN aya VAN d di 0 Evan Evan TLAN IAN 7 2 won wow 35 far Lpofu ipu gw ra M Yd X08 M Yd _ 7
4. TF Probe 1 TF Probe 2 TF Probe 3 TF Probe 4 7 5 Biotin 3 5 Biotin 3 5 Biotin 3 5 Biotin 3 2 Incubate with nuclear extract a TF TF TF TF Jy 5 Biotin 3 5 Biotin 3 5 Biotin 3 5 Biotin 3 3 Separation of protein DNA complexes Spin column Cw u Bind v T pue Wash Vv lt i gt Elute 4 Hybridization Cis 0 Figure 1 Workflow diagram of the Protein DNA Array Page 8 Protein DNA Arrays User Manual Guidelines for Assay Design and Analysis Guidelines for Assay Design and Analysis General Guidelines Assay Procedures Before You Start Preparing Nuclear Extracts 4 Read this user manual and all product inserts before performing the assay Store all reagents at the recommended temperatures Use Panomics Nuclear Extraction Kit for best results Thaw Positive Control and sample nuclear extracts on ice Thaw Probe mix on ice We recommend using Panomics Nuclear Extraction Kit Cat 2002 The minimum protein concentration of nuclear extract should be 1 0 ug ul or greater and we recommend using between 4 10 ug of Nuclear Extract Protein You will need to adjust the amount of water accordingly when preparing the samples with the probe mix When measuring protein concentration we also recommend using the DC Protein assay from Bio Rad Lowry assay with a plate reader or spectrophotometer Preparing the n this Section
5. LI 91 SI vi el cl LE 01 6 8 2 9 9 v C L I v q ureroag joubisunpi jo woog owas zi Andy a gt lt DQ O Au L O T TranSignal Protein DNA Arrays User Manual lt m o A LO TI Uo smoUund oddnsyra Jo iure 10 7599 97 10 poddns poruupey ano espe d sAn m ano pasn 5 41 a44 10 pup sexuanbas snsuasuo ayy oj 249 E Z L 9 siseujueand ur siaquinu pajouap Aat sexuenbas snsuasuod yum S41 404 010 JNO UO SUOIJDLIDA asoy papiAoJd am ejoridouddp asaym pup aj paytsjqnd ueaq any Jj awns 10 sexuenbes snsuasuo Jon uoyd nsun awos 104 0550 10 sjoapuo sp pasn aio pun pamuno ur payods 8 0 Aa g gl vL el cl LL OL 6 8 L 9 9 v C L 2 7 1 Ld8W EN agw LHSVW mar 39 451 pun zH af O f d fodi vagi vam 4951 agro dem punxoid orsip 7 10151 Z 1 Ut E ENISHI ADS ALH ASH gya 3YXOM oray opan LINH LANH yq LLH I 2 0 on i cany ECINH ECANH a IEEE MEE T dm eng led SAH SAH 2 2 ET 14 wa 4v0 DMI MI 16 7 1
6. Observation Possible Cause Recommended Action Uneven Background Substrate is note evenly distributed on the membrane Do not use thin cling wrap materials during the overlay procedure Exposure Time to Short Re detect with substrate that is evenly covering the membrane surface High background Incubation with substrate is too long Incubate with shorter time Non specific protein bound to membrane Increase blocking or washing times Signal is too weak Not enough protein was used in the assay Carefully measure the concentration of the nuclear extract using a Lowry or Bradford assay Protein DNA Arrays User Manual Assay Procedures Observation Possible Cause Recommended Action Don t see difference Confirm concentration of Carefully measure between treated and nuclear extract concentration untreated samples Cells cultured with growth Culture cells overnight factors in serum without serum prior to treatment TF not activated Confirm with EMSA References 1 Chodosh L A Olesen J Hahn S Baldwin A S Guarente L and Sharp PA 1988 A yeast and a human CCAAT binding protein have heterologous subunits that are functionally interchangeable Cell 53 25 35 2 Zeng G L Gao L Xia T Tencomnao T and Yu R 2002 Characterization of the 5 flanking fragment of the human GM3 synthase gene Biochimica et Biophysica Acta 93745
7. 199 6 0 1 489 LvIV9 L VLY9 493 umsohl 493 uso NR L0 INDI L24 ug dH 1B w 2 2 2 di da 2 2 UB EUVVBI zn za 1494 1999 9 339 099 700898 pag z 0 040 1010929 0 z a qan 2 219 7 218 op 130 130 MID WIE gap 7 7 7 7 ggioumque ggioumquo gt Ld Ld 91 4 81 4 41 4N0 41 410 Ay so 1542 587 2 2 c s p p 7 7 w w 389 8D dg a ag agn s wn em ap Z A 9 10pn4 19 2004 _ ojapo Dapo Yd Wd 2 0 7 0 0 7 ugod ugoji we CALM PA cy qvod yodo pay pay We We LIY dad did DY py ay lav aw dW Gl vl EL CL LL Ol 6 8 L 9 9 v C L YNQ U13j014 ubisupa Jo owas Ar Andy X lt DQ A lu Lr TranSignal Protein DNA Arrays User Manual Page 18 LI M lt DQ O A u L O T ulajorg qg Wosdiwoundg joddnsypa jp iure 10 7599 97 10 dnoJ proddns joupa Ino o espe d sAn um 1no pasn S 41 84 10 saduanbas snsuesuo ayy je c Z L siseujueund ur siaquinu pajouap ueaq Aay sexuenbes snsuasuo gt 841 104 S DIJD JNO UO SUOIDLDA asoy papiaoad
8. 2 1 1 3 Yd L Yd GL vL EL CL LL OL 6 8 L 9 G v L YNQ U13j014 w puBieupa Jo woog owas Any lt M O A L O I Page 19 TranSignal Protein DNA Arrays User Manual ulajo14 Duipurg 49 asoudsay 3y Wo syruoupdq uoddnstpa jp 799 97 8 10 dnos poddng Ino ji aspajd s nio INO pesn s y 104 Sauadajad pup seuanbas Snsuasuo ays 249 E z L siseujuaund siaquinu pajouap Aay saxuanbas snsuasuo ejdijjnui s 4 204 010 JNO uo suonna 1 aay am ojpudouddp aym 24401941 aj pay sjgnd uaaq J awns ay 10 sexuanbas snsuasuo ajdijnuu siopo4 uoydsunaj awos 104 0550 104 sp pasn ain perpjAuotq payods suuinjo pup Apiiyoquio bo 6 l E 0 118 0 dtd 7 1 daso 1 01 6 8 N j0xo4 14 dd dYM 1 MIS e 55 05 7 Mdid e a Li a P ams i gt lt A L XVd a v l l jra Ad FI E bo 6 6 lg 0 61 1 LL 91 SI pl l 2 4800 1 d8QqW ccd 1 2 q o Q 4NIH pv prall HAH 80 zt s 08 483 4B r 98 W ag L 39X1 2 983 un IIO 68311 2 LIVAN 7 0 qAW ds L dS ere ele i a
9. the film to obtain numerical data for comparison Protein DNA Arrays User Manual Page 11 Assay Procedures Results amp Analysis TranSignal Arrays give you quick answers when you want to identify those activated transcription factors through comparison of two or more samples Follow these guidelines to analyze your results Stripping Procedure Possible Problems and Recommended Page 12 Solutions 6 1 6 2 6 3 Acquire the images using either x ray film or a chemiluminescence imaging system Adjust the exposure time such that the majority of the positve control spots have equal signal intensity at the same exposure time When comparing mutliple blots ensure that the control spots have a similar intensity Note We do not encourage stripping the array membranes more than two times 7 1 7 2 7 3 7 4 7 5 7 6 Wash membranes in 0 4M NaOH at 45 C for 30 min Wash membranes in 0 2M Tris HCL pH 7 6 0 1X SSC and 0 196 SDS at 45 for 15 min To ensure that stripping was successful follow the standard chemiluminescence detection procedure as described in the user manual After detection wash the membrane twice in 1X TBST with 0 1 SDS at 42 C for 30 min Membranes are ready for hybridization or air dry the membrane at room Store dry membrane protected between paper at room temperature unil ready for further use Recipe for 10X TBST 1M Tris HCL pH 7 5 1 5M NaCl 0 596 Tween 20
10. you will prepare the array membranes for use If you are reusing Membranes membranes that have been previously stripped skip steps and begin from step 1 4 1 1 1 2 1 3 1 4 1 5 Prepare Pre Treatment Buffers and Il see Appendix for recipes and warm the Hybridization Buffer to 42 in a water bath or incubator If you notice a cloudy or soapy appearance for the Hybridization Buffer make sure the particulates are completely dissolved before proceeding It may require overnight heating in a water bath Place each array membrane into a hybridization bottle Be sure to place the membrane in the hybridization bottle such that the spotted oligos face the center of the tube away from the walls Add 25 ml of 1X Pre Treatment Buffer to each bottle and place in a hybridization oven at 45 for 5 minutes Thoroughly decant the solution from each bottle Add 25 ml of 1X Pre Treatment Buffer II to each bottle and place in a hybridization oven at 45 for 10 minutes Decant the solution from each bottle Rinse the entire surface of each membrane with distilled water not provided by filling each bottle approximately half full and swirling the bottle for 30 sec Repeat this rinsing step two additional times and decant water after final rinse To each hybridization bottle add 5 ml of prewarmed Hybridization Buffer provided Place each bottle into the rotating hybridization oven at 42 C for 2 hr If needed you can stop
11. 1 6 3 Miyoshi K Rzucidlo S J Pratt S L and Stice S L 2002 Improvements in Cloning Efficiencies May Be Possible by Increasing Uniformity in Recipient Oocytes and Donor Cells Biology of Reproduction in press 4 Govindarajan B Bai X Cohen C Zhong H Kilroy S Moses M and Arbiser J 2003 Malignant transformation of melanocytes to melanoma by constitutive activation of MAP kinase kinase signaling J Biol Chem Electronically published 1 03 see www jbc org 5 Wheeler M Smutney O Check J Rusyn l Schulte Hermann R and Thurman R 2003 Impaired Ras membrane association and activation in PPARa knockout mice after partial hepatectomy Am J Phsiol Gastrointest Liver Physiol 284 302 312 6 Dignam J D Lebovitz R M and Roeder R G 1983 Accurate transcription initiation by RNA polymerase ll in a soluble extract from isolated mammalian nuclei Nucleic Acids Research 11 1475 1489 Protein DNA Arrays User Manual Page 13 APPENDIX APPENDIX I RECIPES AND INSTRUCTIONS Page 14 75 ml of 1X Pre Treatment Buffer Warm 5X Pre Treatment Buffer to room temperature If precipitate is visible warm gently to fully dissolve solution and allow to cool to room temperature To 60 mL of deionized H O add 15 ml of 5x Pre Treatment Buffer I Mix well 75 ml of 1X Pre Treatment Buffer II Warm 5X Pre Treatment Buffer Il to room temperature If precipitate is visible warm gently to ful
12. A Assay Kit Protein DNA The Protein DNA kit is a highthroughput profiling assay that has the ability to Assay mesaure the activation of multiple TF s from a single sample The cytokine panels for TranSginal Protein DNA kits are available as V Array Name Cat of TF s PDArray MA1210 56 PD Array II MA1211 96 PD Array Ill MA1212 94 PD Array IV MA1213 76 PD Array V MA1214 73 Combo Array MA1215 345 Protein DNA Assay Kit Contents and Handling Conditions Kit Contents and The contains the following components Shelf life of kit is six months from date of Storage receipt under proper storage conditions Page 6 Box 1 Array Membranes and Hybridization Reagents Store at 4 C 9 9 9 9 Protein DNA Array 3 each Spin Column 3 each Spin Column collection Tube 6 each 1X Column Incubation Buffer 2 ml 1X Colunm Wash Buffer 10 ml Hybridization Buffer 15 ml 20X SSC 32 ml 2096 SDS 15 ml Streptavidin HRP conjugate 60 pl 4X Wash Buffer 45 ml Solution 750 ul Solution II 750 ul Solution III 8 ml Box 2 Reaction Kit Store at 20 9 99 9 9 Probe Mix 30 pl 1X Column Elution Buffer 200 ul Bring to room temperature before use Distilled H 0 RNAse DNAse free 500 ul Control Nuclear Extract from HeLa Cells 5 ul 2X Blocking Buffer 30 ml 1X Detection Buffer 60 ml 5X Pre Treatment Buffer 20 ml 5X Pre Treatment Buffer II 20 ml P
13. AAN zw ZW LASAW LASAW raw oem ew uu vlan O CAN CHW zgw caw MI dT ac as a uu 1410 3910 j j j A BE BLLICIE ME Mea MEC MEE MN MN M M ul Mm DW j SHIH guy THH CHH agg SYH S8H SVH SEH T wu 9 LY 1 vg OVD vYIV VIVO eo Evang CIO z L vlv9 1 11 yo 9 9 99 w 7 Q Q foxy M M T ye en p39H za gowa qoe 2309 YYY YM NDH NH mM E LIN z 1 1 D DB AWA ss ag iB SIO oaa ro EM zum 3 1 1 1 1 1 1 ra HO DD 040 BO UEBD HEBDO ro D A 3D zu DON zum ew muss 0 m O M NXN m hp Ap esuodserg esuodsod pay ray Edy dy _ tuepixotjuy juoponuy 3JUF9NY 3UFONY LNAV 4HY INYV YHVI av Xov LL 9 Gl VL el ol LL 01 6 8 L 9 q v Any vy q urejoag ouisuny Jo yowa 4 lt M O QA Lu O TI TranSignal Protein DNA Arrays User Manual Page 16 1 i uoddnsipo jp iure 7599 97 8 10 dnoJ6 poddng piuypa ano aspajd Anm ano pesn s 41 40 saduanbas snsuasuo ayy oj 249 c 7 L siseujueund siequinu payouap uaaq Aayy sexue
14. Protein DNA Arrays Spin Column Separation Version User Manual Cat MA1210 MA1211 MA1212 MA1213 MA1214 amp MA1215 P N 13299 Rev B 032007 Panomics Panomics Inc Protein DNA Array Kit User Manual Copyright Copyright 2006 Panomics Inc All rights reserved Trademarks Hyperfilm and ECL are trademarks of GE Healthcare Inc FluorChem is trademark of Alphalnnotech Inc Citing Protein DNA Arrays in Publications When describing a procedure for publication using this product we would appreciate it if you would refer to it as the Protein DNA Array Kit from Panomics If a paper cites the Protein DNA Array kit and is published in a research journal the lead author s may receive a travel stipend for use at a technology conference or tradeshow by sending a copy of the paper to our technical support group at techsupport panomics com or via fax at 510 818 2610 Disclaimer Panomics Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Panomics Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Contents About the User 5 Who Should Read this
15. ard the flow through Place the Spin Column in a new Spin Column Collection Tube provided Add 600u1 of 1X Column Wash Buffer to the Spin Column and incubate for 10 min on ice Centrifuge column at 7 000 rpm for 30 sec at 4 and discard the flow through Wash the column by adding 600 ul of 1X Column Wash Buffer to the Spin Column and centrifuging at 7 000rpm for 30 sec at 4 Repeat step 3 8 three more times Remove residual Wash Buffer by an additional centrifugation at 10 000 rpm for 30 sec at 4 C Add 60ul of 1X Column Elution Buffer to the center of the Spin Column and incubate at room temperature for 5 min Place the Spin Column in a clean 1 5 ml microcentrifuge tube and centrifuge for 1 minute at 10 000rpm at room temperature Place the microcentrifuge tube containing the collected flow through which contains the eluted probe on ice and use for further steps If needed you can stop at this point and store the eluted probe at 20 overnight In this Section you will hybridize the eluted labeled probe prepared in Section 3 to the array membrane prepared in Section 1 Protein DNA Arrays User Manual 44 42 4 3 4 4 Assay Procedures Denature the eluted probe from step 3 13 by heating it at 95 C for 3 min and quickly chill in ice for 2 min Add the eluted probe to each hybridization bottle and hybridize at 42 overnight in a rotating hybridization oven Decant the hybridization mixture
16. at this point of the protocol and leave the membrane overnight at 42 Protein DNA Arrays User Manual Page 9 Assay Procedures Forming Complexes IsolatingTF Bound Probes Hybridization Page 10 In this Section you will allow the DNA probes to bind to transcription factors from Protein DNA your nuclear extract 2 1 2 2 For each nuclear extract sample combine the following components into a sterile 0 5 ml microcentrifuge tube 2 provided Nuclear extract 1 3 ug ul 5ul TranSignal Probe Mix 10ul dH O RNase DNase free 5yl Total Volume 20yl Note If the protein concentration of your nuclear extract is below the recommended range replace the dH O with additional nuclear extract Mix well by pipeting Incubate samples for 30 min at 15 C In this section you will isolate the protein bound probes from the non bound probes All centrifuge steps should be carried out on a regular benchtop centrifuge at 7 000 rpm at 4 C unless otherwise stated 3 1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 3 10 3 11 3 12 3 13 Wash Spin Column by adding 500 ul of chilled 1X Column Incubation Buffer and centrifuging at 10 000 rpm for 30 sec at 4 or room temp Add 20 ul 1X Column Incubation Buffer to the TF Probe mix from Step 2 2 Transfer all of this mix into the center of the Spin Column Incubate the Spin Column on ice for 30 min Centrifuge column at 7 000 rpm for 30 sec at 4 and disc
17. cumbersome gel mobility shift assays which permit the characterization of only a single TF at a time With these Protein DNA Arrays you can profile the activities of multiple TFs simultaneously These arrays can be used to study TF activation in a variety of biological processes including cell proliferation differentiation transformation apoptosis and drug treatment We currently offer six versions of the TranSignal Protein DNA Array Version for 56 transcription factors TFs Version II for 96 Version Ill for 94 Version IV for 76 Version V for 73 and the Combo Array for 345 TFs These transcription factors are well characterized and widely available in published literature Protein DNA Arrays use a proprietary patent pending technology developed by Panomics for the high throughput analysis of TF activation The procedure is simple and straightforward Figure 1 Three basic steps are involved 1 a set of biotin labeled DNA binding oligonucleotides Probe Mix are preincubated with a nuclear extract of interest to allow the formation of protein DNA complexes 2 the protein DNA complexes are separated from the free probes and 3 the probes in the complexes are then extracted and hybridized to the Protein DNAI Array Each kit includes the reagents for HRP based chemiluminescence detection Protein DNA Arrays User Manual Page 7 Overview of Assay Workflow Overview of Assay Workflow 1 Mixture of pre labeled TF probes T
18. from each hybridization bottle from Step 4 1 and wash each membrane as follows See Appendix I for recipes Add 50 ml of prewarmed Hybridization Wash incubate at 42 C for 20 min in a rotating hybridization oven Decant liquid and repeat wash Add 50 ml of prewarmed Hybridization Wash incubate at 42 C for 20 min a rotating hybridization oven Decant liquid and repeat wash Detection Important Do not allow the membrane to dry during the detection 5 1 5 2 5 3 5 4 5 5 5 6 5 7 5 8 Using forceps carefully remove each membrane from its hybridization bottle and transfer to a clean container containing 20 ml of 1X Blocking Buffer each membrane needs its own container We use a container that is equivalent to the size of a 200 ul pipet tip container 4 5 x 3 5 Block the membrane by incubating at room temperature with the 1X Blocking Buffer for 15 min with gentle shaking Remove 1 ml of the 1X blocking buffer to a clean microcentrifuge tube Dilute 20 ul of the stock Streptavidin HRP conjugate with 1 ml of the 1X Blocking Buffer Vortex the diluted streptavidin HRP and transfer back into the 1X Blocking Buffer from Step 5 2 containing the membrane Add the diluted conjugate to the container making sure not to pour directly on the membrane Continue shaking the membrane for 15 min at room temperature Decant the diluted Streptavidin HRP solution Wash each membrane three times at room tempe
19. jpnur s 4 aut 104 JNO uo SUOILDLIDA asoy anny am ayoudosddo araym pup pay sjgnd uaaq 4j 104 sexuanbas snsuasuo opp uordibsupij awos 104 Q D ID payyods aun Aay you jdaxxe sao apu ayp 9A0QD s 41 IYF SD 54 smoy pappus Apssp ay 10 s o1yuo aaysod sp pasn ain pun payods a n suuinjoo A Any ZL OL SGL l 2 1 Ob 6 8 4 9 g v Z L JUN am o 6 40 40A CoA UA vaL A u zw 398 2 s al u QUAL GIL gms gms sms sms p s gms EWS LPS MS 385 185 ds lds QUAL gms gms pms pms ews 105 ms S 1 6 P tQWs p t QWWS 3g5 pows 3gs pous as a Sud ava Wd Md Ka d a P tQWWs In e aws 3aS pows zas poms as as raazau raaka saaya Saa va ld add NE d i 194 DX a A M di n Lb nh n Ah em UMEN a pw pu oy in e om Ga ma DH VON E xoy W XOW 2AW u 2n s F Lul Lal En ur W u9 Yd 49 VIVO vg fn a Dam mn zum zum am HERE van wm mo um A 301 509 sus SvO 1184 1498 TuS a Y u u Ha 142 i ray cay mss asse LAH ual cuu UO TU 8 m 0 a una 8 vv iw e e m e
20. ly dissolve solution and allow to cool to room temperature To 60 mL of deionized H O add 15 ml of 5x Pre Treatment Buffer Il Mix well 300 ml of 2X SSC 0 5 SDS Hybridization Wash 1 To 262 5 ml of deionized H2O add 30 ml of 20X SSC provided and 7 5 ml of 20 SDS provided Mix well 300 ml of 0 1X SSC 0 596 SDS Hybridization Wash Il To 291 ml of deionized H2O add 1 5 ml of 20X SSC provided and 7 5 ml of 20 SDS provided Mix well 60 ml of 1X Blocking Buffer To 30 ml of deionized H2O add 30 ml of 2X Blocking Buffer provided Mix well and store at 4 C 180 ml of 1X Wash Buffer To 135 ml of deionized H2O add 45 ml of 4X Wash Buffer provided Mix well and store at room temperature Protein DNA Arrays User Manual TranSignal Protein DNA Array Grid TranSignal Protein DNA Array Grid Photocopy this page onto a separate piece of paper or transparency film Note that the notch is on the upper right hand corner of the grid Page 15 lt V O uu Lu ote xl mco Protein DNA Arrays User Manual TranSignal Protein DNA Array Grid Page 16 Protein DNA Arrays User Manual gt X 3 gt Z O lt m O A U L O TI Woy sdwound oddnsypa p jowa 10 7799 97 8 10 dno proddns jpoiuupej INO o asDajd 5 INO pasn s 4 104 sa3u219j81 pun Saduanbas snsuasuo au oj c z 1 siseujuaund sjaquinu pajouap uaaq Aay sexuanbas snsuasuoo ajdi
21. nbes snsuasuo e dijynui SJ 104 S DIJD Ino UO SUOIDLDA asoy pap no d am apoudosddn asaym anyos ay ui pay signd ueaq 4 awns 104 sexuenbes snsuasuo ayd yaw uordiosupij awos 104 Asso ayy 10 0 sp pup vw pamuno payods aio suwnjo so1 e1o i AD1IY Page 17 gt X J gt lt m o A L O TI LI 91 SL Vi el cl LE 01 6 8 2 9 v C L V LINZ vLUNZ n 7 dazx dazx P p d m n PWA PWA UESU UESH l DIE g CIL ea D DI dYS 4US AYS AYS dSV L dS dSV L dS eas eais zas gas LAIS LAIS 00341 ly b zs ay ay Ud Wd pug ang Eis a 4 E 4 4 EA m did 444 diH tVid 652 LAN LUN 7 CIN ZN kA EM E EM varan Yar N s a is us UAW 99A QoAW P L ha y y He qan 03801 LE a oon oron soon snos orou PON omon om mo moo VEM pm e am am m o m o Sh 04 d83 Gr 0rr dti T 7 DD DD qw TAD C830 30 30 Ea Pu w OD p s jin iso beni M 0 WD 200 IM n m dv du 98V 9 98 UA CAY LC LIWY mv 30 30D Lev p Lady Lady
22. o9 9 ug as e om ow HI zw DIU wn am w M feu ew w r mimm 8L ZL 9L SL vl zl Hd OL 6 8 9 g v Z Any YNC U184014 jpu isuns Jo 1 Any gt x S Z o lt M o A U L O TI Page 15 TranSignal Protein DNA Arrays User Manual J gt D lt M O a O TI uo sououpd o uoddnsipey jo 10 7599 97 8 10 dnoa6 Ino espejd soun ano 41 a44 10 saduaiajai pun sexuanbes snsuasuo ayy oj 343 c Z 9 siseujueund srequinu pajouap ueeq Aayy snsuasuo ejdijjnui S 104 S DD ano UO asoy papiAud am ayLdosddo asym pun aanposap ayp pay signd any J awns ayy 10 saJuanbes snsuasuo uoydysuna awos 104 050 10 sj01juo antjsod sp pesn en pun vq payojAunorg payods suwnjo pun smos jj 9 Gl VL el cl LE 01 6 8 L 9 q v G L 012 012 JI Ju 917 au VIZ VIZ 2 40 Ld9X ya 820 0L gnp vo 1 1 2 2 1 1 7 7 Ms ms 5 95 Ws Ws DAS ma guy gu wu wu wu Add u VW BAYA oyyy ma ana DX zaya ad ooed ood MO Mo HX p CN CUN 2 TSYAN STON SCN AIN
23. rature with 20 ml of 1X Wash Buffer each for 8 min Add 20 ml of 1X Detection Buffer to each membrane and incubate at room temperature for 5 min Overlay each blot with 2 5 ml of Working Substrate Solution prepared by mixing the following in order 250 ul Solution with 250 pl Solution Il Briefly vortex and add 2 0 ml Solution Ill Thoroughly mix Using a plastic sheet protector or overhead transparency film do not use thin plastic cling wraps as these wrinkle and lead to uneven detection across the membrane Place each membrane on plastic sheet Then pipet 2 5 ml of the mixed substrate solution onto each membrane and overlay each with a second plastic sheet on a flat surface as quick as possible Ensure that substrate is evenly distributed over the membrane with no air bubbles Incubate at room temperature for 5 min Remove excess substrate by gently applying pressure over the top sheet Using a paper towel remove excess substrate that might be remaining on the surface of the sheets Expose the membranes using either Hyperfilm ECL 2 10 min or a chemiluminescence imaging system 5 15 min such as the FluorChem imager from Alpha Innotech Corp In either case we recommend that you try several different exposure times Obtain quantitative analysis if desired If you are using a chemiluminescence imaging system follow the instructions that are provided with that system s software If you are using Hyperfilm ECL you will need to scan
24. rotein DNA Arrays User Manual Additional Materials Required Materials and Euipment Introduction Princicple of the Technology Protein DNA Assay Kit Contents and Handling Conditions Panomics Nuclear Extraction Kit AY2002 Deionized H 0 0 5 ml and 1 5ml microfuge tubes Pipetman and tips Microcentrifuge Hybridization oven and bottles Plastic containers 4 5 x 3 5 equivalent to the size of a 200 ul pipete tip box Shaker Plastic sheet protectors Hyperfilm ECL Amersham Cat RPN3114K Chemiluminescent Imaging system FluorChem from Alpha Innotech 9 9 9 94 9 Eukaryotic gene expression is regulated by a group of proteins called Transcription Factors TFs By interacting with specific DNA Binding elements present in the promoters of certain genes TFs modulate the frequency of transcriptional initiation The expression or activity of TFs may be regulated in a cell type tissue specific or cell cycle dependent manner Regulation can also be mediated by interactions with other proteins Through different combinations of these regulatory mechanisms eukaryotes are able to elicit a myriad of different gene expression patterns The key to a full understanding of how gene expression is regulated is the analysis of the biochemical activity of TFs Panomics Protein DNA Arrays simplify the functional analysis of eukaryotic TFs Our array based technology is a significant improvement over
25. s EMIS HE dd 2X m 2415 zum vn E 145 DE apy 182900 DU 2 WW qab w um LIZW 1 1 M 2 1 S HP Sh 0b 1 ml id dg 2 0 01 6 qi 1 1 opoy LYLY9 arano 90 W aygy gy 120 cm wa 8 9 elo 6 8 1 9 S p Z 1 S XVd D D evad SH ar Lm 2 ZH cu 1 451 E 3 1 c EY 1 A 419 10 10 oS ot 1015 T gt lt uw O L o lt lt Ci E VLV9 31 453 e ex CX e lid e x S 31 01 ya CVIV9 Ez _ CN IL a IM IM xow Aw dQ a 1 19 1 483 IM gt 25 FIVE o N r Wd 2 e E eal s o C a r esm vn fona i mo aa 07 xoq M 1 a 2 Um WSI g qhu xog IW Z l m jo iunii DNA Arrays User Manual 1 Protein TranSigna Page 20
Download Pdf Manuals
Related Search