TF Interaction Arrays
Contents
1. og J aN LEW ZH LIST OL oew Oc Voc uc an Ps PPP uS sump sem dea de NH EHIH CHAH HAH HH SAH LO salva vo vto cto eto cuo curo svo svo 909 209 PI pwy poney x E ee a m p E HA Dip pg 28 vaa 1 EA EMO 136 0 OO ue andy ands D THD HD UD CIN queue 0941 09410 WI nw hoy roni WINY INY Aw AW L ee fl 9 Si ge d Hu og 6 8 9 6 been spotted These spots are intended for alignment Note that the notch is at the Figure 3 Schematic diagram of the Interaction Array Il The dark grey columns top right hand corner along the right and bottom sides of the array indicate where biotinylated DNA has Interaction Arrays User Manual OL sn g um HIE 01 S asas lasas st as asv tas CIS CIS GS HIS LS teu Pc TU ay2. mpm m pow we Rp 5 gs pus S 0 Lata 0 5 89 Da Iaou 980 pw m om on on m sw per p er we su sur noc on on va rn m m pi forn een re a nn e popup m www wr oar e e sw pus ver er sw pw purpura We qm D 4 47 eug qw Una Una qN3911 diagram of the Interation Array I The genes on the array are tic spotted in duplicate the first row is DNA spotted normally the second row is DNA diluted 1 10 The dark grey columns along the right and bottom sides of the array indicate where biotinylated DNA has been spotted These spots are intended for alignment Note that the notch is at the top right hand corner Figure 2 Schema Interaction Arrays User Manual Page 18 APPENDIX B Schematic diagram of the Interaction Array Il fol QZ QZ MZ HX La vni 56 Page 19 AMS MS US US Lis Cole Code Add Pd 0910 QXOY 9XOY 9x04 pxog gxog O04 10 EIN lean
3. Il and are provided in Appendices B C and D respectively By Interaction Arrays User Manual Page 3 Introduction Page 4 p300 SRC combining al of these versions you can profile the interactions of 244 unique TFs However the TF TF Interaction Array system can be easily expanded to include up to 2 000 consensus sequences To initiate transcription activated TFs must either bind to their cis element sequences or interact with other TFs To connect the TFs to one another and to the basal transcriptional machinery secondary proteins called cofactors are usually required The interactions between TFs cofactors and the components of the tran scriptional machinery create a complex multidimensional transcription network One group of proteins involved in this network is the p300 CREB binding protein CBP family Originally discovered through their binding to the adenoviral E1A and CREB proteins p300 and CBP are now known to be potent transcriptional co activa tors More than 30 transcription factors p53 ER HIF1 Stat1 E2F 1 and Jun to name a few have been identified that interact with p300 CBP 1 3 To regulate TFs p300 CBP integrates into the transcriptional apparatus becoming a physical link between TFs cofactors and the basal transcription machinery This interaction occurs through three distinct mechanisms p300 CBP acts as a bridge to connect TFs directly to the basal transcription machinery 3 5 p300 CBP for
4. m Md Wad png flan handel Una anda 706006 444 dead Sod Interaction Arrays User Manual EN AAN CN CIN ua a uy rRNA ON DUAN PUSAN KEW UAW IAW 0 HW FEN zw ZW Oc DTE ERIT Kor os pn OH sod c VESNH mu mw qu PP INAH app JB RC OD CLR oa 0 0B DD DD 9 BD CDD 0 0 til DD 09 igi DD 28200 0 01 90 00 GU GUI Schematic diagram of the Interaction Array APPENDIX B n amp da uo 6 8 6 1 Figure 4 Schematic diagram of the Interaction Array The dark grey columns top right hand corner along the right and bottom sides of the array indicate where biotinylated DNA has been spotted These spots are intended for alignment Note that the notch is at the Page 20 APPENDIX E APPENDIX E TF TF Protein Photocopy this page onto separate piece of paper or transparency film Note that the Interaction Array notch is closest to A24 Grid gt gt 2 gt O SS gt 6
5. 1983 Accurate transcription initiation by RNA polymerase ll in a soluble extract from isolated mammalian nuclei Nucleic Acids Research 11 1475 1489 p300 TF 1 Janknecht R and Hunter T 1996 Transcription control versatile molecular glue Curr Biol 6 22 23 2 Janknecht R and Hunter T 1996 MAP kinase dependent transcriptional coacti its cofactor Biochem Biophys Res Commun vation by Elk 1 228 831 837 3 Shikama N Lyon J and La Thangue N B 1997 The p300 CBP family integrat ing signals with transcription factors and chromatin Trends Cell Biol 7 230 236 4 Goodman R H and Smolik S 2000 CBP p300 in cell growth transformation Interaction Arrays User Manual Page 15 Materials Page 16 and development Genes Dev 14 1553 1577 5 Chan H M and La Thangue N B 2001 p300 CBP proteins HAT s for transcrip tional bridges and scaffolds J Cell Science 114 2363 2373 6 Xu L Glass C K and Rosenfeld M G 1999 Co activator and corepressor complexes in nuclear receptor function Curr Opin Genet Dev 9 140 147 7 Chan H M and La Thangue N B 2001 p300 CBP proteins HAT s for transcrip tional bridges and scaffolds J Cell Science 114 2363 2373 8 Ogryzko V V Schiltz R L Russanova V et al 1996 The transcriptional co acti vators p300 and CBP are histone acetyltransferases Cell 87 1107 1112 9 Korzus E Torchia J Rose D W et al 1998 Transcription factor s
6. Adjust the exposure time such that the biotin spots along the right and bottom sides of the membrane have equal signal intensity Compare the images between negative control normal IgG and experimental All the spots that appear in your experimental should be positive in contrast the negative control should not have any spots If the image of the negative control has some weak spots then you ll need to check whether the same spots also appear in the experimental If so the spots in the experimental may represent false positives To confirm your results perform co immunoprecipita tion or super gel shift assays Toubleshooting Guide Problem Cause Recommendation Uneven background Substrate is not evenly distributed on the membrane Do not use thin cling wrap materials during the overlay procedure Re detect with substrate that is evenly covering the membrane surface High Background Incubation with substrate is too long Expose longer 10 to 15 min Incubation should not exceed 5 min Signal is too weak The yield of recovered DNA probe is low Confim using control nuclear extract provided Check the concentration of nuclear extract No Signal The antibody may be not suitable for IP Test the antibody before applying to the array References TF TF SRC TF PPARalpha TF NFkB p50 TF GRalpha TF 1 Dignam J D Lebovitz R M and Roeder R G
7. has shown that SRC1 can also bind to transcription factors that are not nuclear receptors such as TBP and TFIIB These findings suggest that SRC1 is a bridging coactivator that connects nuclear receptors to other transcription factors and an integral part of the regulation of hormone induced cellular response Tradi tionally protein interactions have been studied by co immunoprecipitation and super gel shift But because these methods are notoriously time consuming and inef ficient they are not conducive to mapping the network of interactions between the TFs and the SRC1 cofactor The SRC1 TF Interaction Arrays enable you to determine how SRC1 interacts with multiple TFs all in one experiment This array based sys tem for detecting protein interactions is based on Panomics proprietary TF TF Inter action Array technology We currently offer two versions of the SRC1 TF Protein Interaction Array Version 1 is spotted with 54 different consensus sequences Version Il is spotted with 96 different consensus sequences and Version lll is spotted with 94 different consensus sequences Schematic Diagrams of Versions Il and IIl are provided in Appendices B C and D respectively By using all three versions you can profile the interac tions of SRC1 with 244 unique TFs PPARalpha Peroxisome Proliferator Activated Receptors or PPARs are nuclear proteins that compose a subset of the steroid receptor super family These proteins were first identi
8. sure to pipet off as much fluid as possible in this step 6 4 Repeat Step 6 6 1 3 three times 7 Elute probe from beads 7 1 Preheat heat block to 100 C Heat the Elution Buffer to dissolve precipitates before use 7 2 Add 60 ul of 1X IP Elution Buffer to tube containing beads Gently mix 7 3 Incubate at 100 C for 5 min 7 4 Transfer to ice immediately Keep on ice for 2 min 7 5 Spin tube in a microcentrifuge at 2 000 rpm for 5 sec 7 6 Place tube in magnetic stand for 2 min to collect beads on ice i e place magnetic stand on ice with tubes 7 7 Important Transfer the fluid to a clean tube and place tube on ice This is your eluted probe The beads can be discarded If you do not plan to use the probe immediately store the probe at 20 C Prior to use thaw probe and repeat steps 6 7 3 and 6 7 4 to ensure complete denaturation of double stranded DNA Hybridization in this Section you will hybridize the eluted probe prepared in Section 6 to the array membrane 1 Place each array membrane into a hybridization bottle To wet the membrane fill bottle with deionized H O Then carefully decant water 2 To each hybridization bottle that contains an array membrane add 3 5 ml of prewarmed Hybridization Buffer provided Place each bottle in the hybridiza tion oven at 42 C for 2 hr 3 Add the eluted probe from step 6 7 7 to each hybridization bottle and hybridize at 42 overnight 4 Decant the hybridization m
9. 2 nO Interaction Arrays User Manual Page 21 APPENDIX F APPENDIX F Stripping Procedure for Page 22 Arrays Note We do not encourage stripping the array membranes more than two times Procedure 1 2 Wash membranes in 0 4M NaOH at 45 for 30 Wash membranes in 0 2M Tris HCL pH 7 6 0 1X SSC and 0 1 SDS at 45 for 15 min To ensure that stripping was successful run it through the standard chemilumi nescence detection procedure as described in this user manual After detection wash the membrane in 1X Washing Buffer at 42 C for 30 min Membranes are ready for hybridization or dry the membrane in an 80 incuba tor for later use Interaction Arrays User Manual Contacting Panomics Contacting Panomics For ordering information or technical support contact the appropriate resource provided below according to your geographical location Location U S Corporate Headquarters Address Panomics Inc 6519 Dumbarton Circle Fremont CA 94555 USA Telephone 1 510 818 2600 FAX 1 510 818 2610 Email info panomics com Technical Support techsupport panomics com or 1 877 726 6642 option 3 Ordering Information orders panomics com Location European Headquarters Address Panomics Srl Via Sardegna 1 20060 Vignate Milano Italy Telephone 39 02 95 360 250 FAX 39 02 95 360 992 Email info_europe panomics c
10. A If you plan to proceed directly to overnight hybridization we recommend that you first prehybridize your membranes see Section 7 1 7 2 before beginning with Section 6 Estimated time 2 3 hours Using a specific antibody against your TF of interest you will pull out that TF and any associated TFs along with corresponding cis elements Wash away free cis ele ments and nonspecific binding proteins then elute the cis elements for use as probe NOTE Antibodies bind with different affinities to protein A or protein G We recom mend that you use the chart below to choose the best affinity Dynabeads for your specific antibody Note Do not use agarose or sepharose beads as these bind non specifically to DNA providing false positive results Ig origin Dynabeads mouse 1961 protein G mouse 192 1926 protein A mouse IgG3 protein G rabbit IgG1 protein A or protein G goat IgG protein G Add 0 196 BSA to 1X IP Dilution Buffer and 1X IP Wash Buffer for protein stability Interaction Arrays User Manual Page 11 Materials 1 Tothe tube prepared in step 5 add IP Dilution Buffer 200 ul Antibody for your TF 200 ug ml 10 ul Note The amount of antibody for immunoprecipitation is variable adjust the amount accordingly For your negative control replace antibody with normal IgG For your positive control replace antibody with p53 Goat Polyclonal IgG 2 Mix well by gent
11. Detection Buffer to each membrane and incubate for 5 min at room temperature 6 To prepare the substrate solution mix 1 ml Substrate Solution and 1 ml Sub strate Solution Add 1 ml Substrate Solution III Mix well 7 Using a plastic sheet protector overhead transparency film or plastic wrap whichever is readily available place each membrane on a plastic sheet Then pipet 3 ml of substrate solution onto each membrane and overlay each with a second plastic sheet Ensure that substrate is evenly distributed over the mem brane with no air bubbles Incubate at room temperature for 5 min 8 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 or a chemilu minescence imaging system such as the FluorChem imager from Alpha Inno tech Corp In either case we recommend that you try several different exposures of varying lengths of time e g 2 5 min Overexposure of your blot may result in excessive background Interaction Arrays User Manual Materials Results amp Analysis main advantage of the TF TF Interaction Array is that you can detect the interac tion of a protein with multiple transcription factors simultaneously Follow these guidelines to analyze your results 1 Acquire the images using either x ray film or a chemiluminescent imaging sys tem
12. NA from protein wash away protein l E hybridize to TranSignal Array Differential signals correspond to different patterns of TF interaction Figure 1 Schematic flow chart of the TF Interaction Array procedure Page 8 Interaction Arrays User Manual Materials Materials Provided Materials Box 1 Array Membranes amp Hybridization Reagents Interaction Array either p300 TF SRC1 TF 3 each PPARalpha TF NFkB p50 TF or GRalpha TF Hybridization Buffer 15 ml Substrate Solution I 3 ml Substrate Solution Il 3 ml Substrate Solution 5ml 1000x Streptavidin HRP Conjugate 60 ul 20X SSC 32 ml 2096 SDS 15 ml 4X Wash Buffer 45 ml 10X Detection Buffer 25 ml Distilled H O RNase amp DNase free 300 ul Box 2 Reaction Kit TF TF Probe Mix 60 ul Poly d I C 50 ul Control Nuclear Extract 10 ul 2X Blocking Buffer 30 ml 5X Binding Buffer 100 ul 5X IP Wash Buffer 2x1 5ml 1X IP Dilution Buffer 1 5 ml 1X IP Elution Buffer 0 5 ml These reagents are provided as stock solutions See Appendix A for instructions on how to dilute these solutions buffers Depending on which kit you purchased you will also receive p53 Goat Polyclonal IgG TF TF 10 ul 200 pg ml p300 Antibody rabbit polyclonal p300 TF 45 ul 200 pg ml SRC1 Antibody rabbit polyclonal SRC1 TF 45 ul 200 pg ml PPARa Antibody either Pol
13. TF Interaction Arrays User Manual Cat 4 MA5000 MA5063 Panomics Panomics Inc p300 TF Interaction Array User Manual Copyright Copyright 2008 Panomics Inc All rights reserved Trademarks Hyperfilm and ECL are trademarks of GE Healthcare Inc FluorChem is a registered trademark of Alphalnnotech Inc All other trademarks are of their respective owners 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 p300 TF Interaction Array Kit from Panomics If a paper cites our product 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 Introduction Introduction Introduction The human genome encodes approximately 35 000 genes While some of these called housekeeping genes are constitutively expressed most are n
14. ed to the nucleus target genes regulates the transcription of various target genes Just a few examples of target genes include interferon b various cytokines genes encoding cell adhe sion molecules and p53 The expression of NFkB target genes depends not only on the activity of itself but also on a number of other factors including NFkB s interaction with other tran Interaction Arrays User Manual Introduction scription factors Thus NFkB target genes might not be induced even when is active Furthermore the pattern of NFkB target genes will be different under different physiological conditions By profiling the expression pattern of target genes we can begin to elucidate the NFkB linked functions of those genes Both NFkB p50 TF and p65 TF Interaction Arrays come in three versions Ver Sion is spotted with 54 different consensus sequences Version Il is spotted with 96 different consensus sequences and Version is spotted with 94 different consensus sequences Schematic Diagrams of Versions Il and IIl are provided in Appendices B C and D respectively By using all three versions you can profile the interac tions of NFkB with 244 unique TFs GRalpha Glucocorticoid receptor GR is a 94 kDa intracellular protein which belongs to the family of steroid hormone receptors GR mediates the effects of glucocorticoids and functions as a ligand activa
15. expres sion In this manner nuclear receptors play a significant role in critical physiological processes such as development reproduction and metabolism Like most transcription factors nuclear receptors depend on cofactors or coregula tor proteins to regulate gene expression Cofactors can act as a bridge to bring Interaction Arrays User Manual Introduction nuclear receptors into close contact with chromatin or the basal transcription machinery to facilitate transcription Alternatively cofactors can form multi protein complexes with nuclear receptors to alter their ability to bind DNA Cofactors with negative affects on transcription are called corepressors whereas cofactors with positive effects on transcription are called coactivators Steroid and nuclear hormone receptors are coactivated by interaction with a cofac tor dubbed the steroid receptor coactivator SRC There are three splicing variants of this particular coactivator While the functionality of each isoform differs the SRC family shares histone acetyltransferase activity and characteristic structural ele ments which include a basic helix loop helix motif a PAS domain and a nuclear localization signal The first SRC to be cloned and identified was steroid receptor coactivator 1 SRC1 This 160 kDa cofactor enhances the transcriptional activity of the nuclear receptors for estrogen progesterone glucocoricoid thyroid hormone and retinoid X hormone Research
16. f critical biological processes PPARa is involved in the transcriptional regulation of enzymes that break down fatty acids PPARg promotes the creation of adipocytes the fat cells that con trol lipid homeostasis and glucose metabolism Disruption of PPAR function has been implicated in the development of several chronic diseases including diabetes and cancer More than 15 years after its discovery nuclear factor kB still commands the interest of researchers in diverse fields This is due to a variety of factors including NFkB s unusual and rapid regulation the wide range of genes that it controls its cen tral role in immunological processes and its involvement in various diseases Regulation of NFKB is a family of structurally and functionally related proteins that regulate the tran scription of a wide variety of genes The NFkB proteins which include p65 Rel c Rel p105 50 NFkB1 p100 52 NFkB2 and p68 Rel B interact to form homodimers or heterodimers that bind DNA with different specificities Uninduced NFkB is sequestered in the cytoplasm and is bound by IkBs proteins that specifically inhibit NFKB family members NFkBs can be activated by a number of agents including lipopolysaccharide phorbol esters IL 1 TNFa protein synthesis inhibitors and reactive oxygen intermediates Treatment with these agents leads to the phosphorylation and degradation of and subsequent translocation of induc
17. fied by their ability to bind peroxisome proliferators fatty acid like molecules that modulate hepatic lipid metabolism It is now well established that PPARs are ligand dependent TFs and are activated by PPs endogenous fatty acids and other metabolites When bound to their cognate ligands PPARs form a heterodimer with another steroid receptor retinoid receptor X RXR This protein complex then induces the expression of metabolism related genes through binding to specific DNA response elements PPARs are also known to interact with other TFs including Sp1 and Smad4 to regu late transcription Through these mechanisms the PPAR family of nuclear receptors Interaction Arrays User Manual Page 5 Introduction Page 6 NFkB p50 is responsible for the cellular response to excess or shortage of dietary factors an essential process for maintaining homeostasis Three isoforms of PPAR exist a b and g each playing a separate role in systemic metabolism The localization of each protein is illustrative of this diversity of function PPARa is found predominantly in the liver heart kidney as well as other tissues where metabolism is elevated PPARg on the other hand is found only in adipose tissue and tissues related to the immune system Of the three PPARb is the most ubiquitously expressed Despite its abundance the physiological role of PPARb is largely unknown PPARa and PPARg on the other hand have been identified as regulators o
18. ixture from each hybridization bottle and wash each membrane 4 1 Add 50 ml of prewarmed Hybridization Wash incubate at 48 C for 20 min in a rotating hybridization oven Decant liquid and repeat wash 4 2 Add 50 ml of prewarmed Hybridization Wash incubate at 48 C for 20 min in a rotating hybridization oven Decant liquid and repeat wash See Appendix A for recipes Interaction Arrays User Manual Page 13 Materials Page 14 Detection IMPORTANT Do not allow the membrane to dry during the detection Dilute all solu tions before use see Appendix A Pre warm the 1X Blocking Solution and 1X Wash Buffer at 37 50 C until all particulates are dissolved 1 Using forceps carefully remove each membrane from its hybridization bottle and transfer to a new 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 ml pipet tip container 4 5 x 3 5 2 Incubate at room temperature for 30 min with gentle shaking 3 Transfer 1 ml of Blocking Buffer from each membrane container to a fresh 1 5 ml tube To each add 20 ul of 1000x Streptavidin HRP Conjugate and mix well Return each mixture to the appropriate container and incubate at room temperature for 20 min 4 Decant the Blocking Solution containing the antibody Wash each membrane three times at room temperature with 20 ml of 1X Wash Buffer each 5 min 5 Add 20 ml of 1X
19. ly tapping the tube Place sample on rocking platform and rock gently for 90 min at 4 C 3 Wash Dynabeads 3 1 Resuspend the Dynabeads thoroughly in vial 3 2 Transfer 75 yl of the Dynabead solution to 1 5 ml microcentrifuge tube 3 3 Place tubes in magnetic stand for one minute beads will precipitate out Pipet off fluid and discard 3 4 Equilibrate the beads by resuspending them in 200 yl of 1X IP Dilution Buffer 3 5 Place tube in magnetic stand for one minute Carefully remove and discard the supernatant without disturbing the beads attached to the wall of the tube 4 Transfer the DNA protein antibody complex prepared in Step 6 2 to the tube containing the washed beads Rock gently on the rocking platform at 4 C for 1 hr 5 Collect beads 5 1 Briefly spin tube in a microcentrifuge at 2 000 rpm for 2 sec 5 2 Place tube in magnetic stand for 2 min to collect beads Keeping tubes on magnetic stand pipet off fluid and discard do not disturb pellet 6 Wash beads with 1X IP Wash Buffer 6 1 Add 400 yl of pre chilled 1X IP Wash Buffer to tube containing beads Com pletely resuspend the beads by gently tapping the tube IMPORTANT Gently invert the tube four times to clean the sides of the tube 6 2 Spin the tube in a microcentrifuge at 2 000 rpm for 2 sec 6 3 Place the tube in magnetic stand for 2 min to collect beads Pipet off fluid Page 12 Interaction Arrays User Manual Materials and discard Important Be
20. ms a scaffold for the assembly of multi protein cofactor complexes 5 6 and p300 CBP increases access to the DNA by modifying chromatin structure with histone acetyl transferase HAT activity 5 7 8 The distribution of p300 CBP among TFs is believed to be signal dependent 5 9 By responding to activated signal pathways p300 CBP mediates a wide array of cel lular processes including proliferation differentiation and apoptosis 1 3 5 Muta tions in p300 CBP genes have even been found in some human tumors suggesting a tumor suppressor functionality 10 10 To fully understand the many physiological roles of p300 CBP and the relationships between signaling pathways and TFs the intricate network of p300 CBP interactions must be dissected We currently offer two versions of the p300 TF Protein Interaction Array Version is spotted with 54 different consensus sequences Version Il is spotted with 96 different consensus sequences Schematic Diagrams of Versions and Il are provided in Appendices B and C respectively By combining both versions you can profile the interactions of p300 with 150 unique TFs Hormones vitamins and other metabolites exert their cellular effects through binding to a specialized class of transcription factors known as nuclear receptors By inter acting directly with DNA response elements as either homo or heterodimers nuclear receptors transduce the effects of their ligands into the regulation of gene
21. om Technical Support techsupport_europe panomics com Ordering Information order_europe panomics com Location Asia Pacific Headquarters Address Panomics Inc 16F Gemdale Plaza Tower A No 91 Jianguo Road Beijing 100022 PR China Telephone 86 10 59208157 FAX 86 10 592081 11 Email info_asia panomics com Technical Support techsupport_asia panomics com Ordering Information order_asia panomics com For an updated list of FAQs and product support literature visit our website at www panomics com Interaction Arrays User Manual Page 23
22. ot Non house keeping genes are expressed in a particular type of cell under specific environmental conditions or at a certain stage of development The expression of such genes must be tightly regulated Gene expression is regulated by a group of proteins known as transcription factors TFs An estimated 2 000 TFs are at work in any given human cell By binding to spe cific DNA cis elements each TF contributes a different functional role in gene expres sion Just as TFs regulate gene expression the TFs themselves are regulated in a number of ways Known as combinatorial regulation these different levels of regulation allow gene expression to be tightly controlled TFs may be regulated by protein modifica tion such as phosphorylation protein translocation and or protein interaction In the latter category TFs may interact with upregulators or downregulators in the basal transcription machinery or with other transcription factors TF TF A novel method for detecting TF TF interactions Traditionally TF TF interactions have been studied by co immunoprecipitation and super gel shift But because these methods are notoriously time consuming and inef ficient they are not conducive to mapping the network of TF TF interactions TF TF Interaction Arrays enable you to determine how a particular TF interacts with multiple other TFs all in one experiment This technology is based on the non cova lent binding of DNA binding sequences to TF
23. pecific recruitment of co activators and their acetyltransferase functions Science 279 703 707 10 Moran E 1993 DNA tumour virus transforming proteins and the cell cycle Curr Opin Genet Dev 3 63 70 11 Giles R H Peters D J M and Breuning M H 1998 Conjunction dysfunction CBP p300 in human disease Trends Genet 14 178 183 Interaction Arrays User Manual APPENDIX A APPENDIX A Recipes amp SECTION 6 instructions for diluting stock solutions 15 ml of 1X IP Wash Buffer To 12 ml of deionized add 3 ml of 5X IP Wash Buffer provided SECTION 7 300 ml of 2X SSC 0 5 SDS Hybridization Wash 1 To 262 5 ml of deionized H O add 30 ml of 20X SSC provided and 7 5 ml of 20 SDS provided Mix well 300 ml of 0 1X SSC 0 5 SDS Hybridization Wash 11 To 291ml of deionized add 1 5 ml of 20X SSC provided and 7 5 ml of 2096 SDS provided Mix well SECTION 8 60 ml of 1X Blocking Buffer To 30 ml of deionized H O add 30 ml of 2X Blocking Buffer provided Mix well and store at 4 C 200 ml of 1X Wash Buffer To 150 ml of deionized H O add 50 ml of 4X Wash Buffer provided Mix well 100 ml of 1X Detection Buffer To 90 ml of deionized H O add 10 ml of 10X Detection Buffer provided Mix well Interaction Arrays User Manual Page 17 APPENDIX B APPENDIX B Schematic diagram of the Interaction Array Le ara pp m on pn e os m s p
24. proteins After precipitation with spe cific antibodies against a target TF the co precipitated TFs are determined by array analysis of attached DNA sequences The advantage of this technology is its capabil ity of high throughput analyzing interactions of native TF proteins in a given sample Figure 1 illustrates how this simple procedure works First incubate your nuclear extract with the provided set of biotin labeled double stranded oligonucleotide probes which represent a known library of cis elements During the incubation step these probes bind to their corresponding TFs in your nuclear extract The next step is immunoprecipitation using a specific antibody against your TF of interest pull out that TF and associated TFs along with corresponding cis elements Next wash away free cis an extracellular stimulus binds to a cell surface receptor Binding trig gers a signaling cascade which migrates through the cytoplasm and into the nucleus Once transmitted to the nucleus the signal regulates transcription factors which turn on and off genes that elements and nonspecific binding proteins Finally elute the cis elements and hybridize them to the Protein DNA Array membrane We currently offer three versions of the TF TF Interaction Array Version is spotted with 54 different consensus sequences Version II is spotted with 96 different consen sus sequences and Version Ill is spotted with 94 different consensus sequences Maps of Versions
25. sions 150 mm x 35 mm diameter tubes Plastic containers 4 5 x 3 5 equivalent to the size of a container for 200 ml pipet tips Heating block or water bath Shaker Plastic sheet protectors or overhead transparencies Hyperfilm ECL Amersham Cat RPN3114K OR Chemiluminescence imaging system e g FluorChem from Alpha Innotech Corp Preparing Nuclear Nuclear extracts can be prepared using the method described by Dignam et al 1 Extract from Cells Alternatively you can use a commercially available kit such as Panomics Nuclear Page 10 Interaction Arrays User Manual Materials Preparing 1 Foreach nuclear extract sample combine the following components into a ster TF Bound DNA ile 1 5 ml microcentrifuge tube Prepare an additional sample for your negative control Nuclear Extract 10 ul Poly d I C 10 ul TF TF Probe Mix 10 pl 5X Binding Buffer 15 ul RNase DNase free 30 ul Total Volume 75 yl Use Control Nuclear Extract for positive control We recommend using 30 100 Ug of nuclear extract per reaction Adjust the volume of dH O based on the concentration of your nuclear extract 2 Mix well by pipeting 3 Incubate samples at 15 C for 30 min Afterwards incubate on ice for an addi tional 30 min Immuno precipitation amp BEFORE YOU START Chill all solutions to 4 prior to use Dilute solutions accord Elution ingly see Appendix
26. ted transcription factor playing an important role in the development metabolism and neurobiology of programmed cell death and anti inflammation In the absence of glucocorticoid GR is bound by HSP 90 and localizes in the cytoplasm Upon glucocorticoid binding GR becomes hyperphos phorylated dissociates from Hsp90 and translocates into the nucleus In the nucleus GR binds to a specific response element in the promoter region of a target gene and regulates gene expression These responsive genes make up around 20 96 of the human genome As corresponding ligands glucocorticoids are among the most widely prescribed class of drugs in the world GR TF Protein Interaction Arrays come in three versions Version is spotted with 54 different consensus sequences Version ll is spotted with 96 different consensus sequences and Version Ill is spotted with 94 different consensus sequences Sche matic Diagrams of Versions Il and Ill are provided in Appendices B C and D respectively By using all three versions you can profile the interactions of GR with 244 unique TFs Interaction Arrays User Manual Page 7 Introduction add TranSignal probe mix to your nuclear extracts control extract experimental extract a DNA protein _ T complexes ie en Bc add antibody against 4 TF of interest SR ae 7 immunoprecipitate using magnetic beads e wash away unbound protein and probe separate D
27. yclonal OR Monoclonal PPARalpha TF 45 ul 200 pg ml NFkB p50 Antibody either Polyclonal OR Monoclonal NFkB p50 TF 45 ul 200 pg ml GR Antibody Polyclonal Rabbit IgG GRalpha TF 45 ul 200 pg ml Interaction Arrays User Manual Page 9 Materials Additional Materials Required Reagents amp Solutions Nuclear Extraction kit e g Panomics Nuclear Extraction Kit Cat AY2002 Hybridization Wash I 2X 55 0 5 SDS Hybridization Wash II 0 1X 55 0 5 SDS Deionized H2O Antibody directed against your TF of interest Optional Normal IgG Antibody for your negative control during immunoprecipitation Choose a normal IgG antibody that is specific to your antibody of choice See Appendix A for recipes Materials and Equipment Dynabeads with either protein A Dynal Biotech Cats 100 01 or protein G Dynal Biotech Cats 100 03 depending on antibody used for immunoprecipitation Please do not use agarose or sepharose beads as these bind non specifically to the DNA probes providing false positive results Magnetic separation stand Promega Cat 25332 0 5 ml and 1 5 ml tubes Pipetman and tips Microcentrifuge Hybridization oven and bottles T Stratagene Cat 401030 T Note skirted centrifuge tubes with screw caps may be used in place of the hybridization bottles VWR Cat 4 21008 480 Hybridization bottle dimen
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