TranSignalTM TF Protein Array
Contents
1. 43U402 PUDY Y 814 do AY 1D S1 YIJOU ay WY aJON uMaUUsYY 40f papuajul aup sjods asayy aubiquaui ayy fo pz gg uuinjor apis 14811 ay Suopp aypaydnp u1 pup q Mod wonoq ay Suopp payods uaaq Spy 4ayADU JJH aiwoydnp u1 payods asp Ap ssy ay uo sujajoid ay Avsd py UldjOdd AJ VUSIG UDALL ay fo wDAsSDIp JyDMaYyIS For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 16 TranSignal TF Protein Array TranSignal TF Protein Array 17 APPENDIX D Schematic diagram of the APPENDIX E Typical Results of the TranSignal TF TranSignal TF Protein Array Version IV Protein Array when screening protein protein interactions A Purified p53 Protein ss Gg ee J se A _ ee ee ee 00 B s ee a C 2 3 ee ee ee e D ee SC oeOeroccesereceeeses 12 345 6 7 8 9101112131415 1617 18 19 20 21 22 23 24 B Bacterial Lysate containing p53 ee ee ee A z 7 Ooo ee p A eereree C D eeereee ee o D ee E ecerseeesereegcergegretee FF 12 345 6 7 8 9101112131415 1617 18 19 20 21 22 23 24 been spotted along the bottom row E and in duplicate along the right side column 23 24 of the membrane2. proteins and their complimentory cis binding element This means that by simply producing a biotinylated version of your promoter region of interest you can asses which transcription factors arrayed on the membrane have the potential to bind to this region Once these have been identified it starts to give clues about which transcription factors and their associated signaling pathways might be regulating the expression of your gene of interest Biotinylated promoter sequence Incubate with TranSignal TF Protein Array membrane Chemiluminescent detection Signal strength corresponds to strength of protein DNA interaction Figure 2 Flow chart of the TranSignal TF Protein Array DNA Promoter interaction For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 5 TranSignal TF Protein Array 2 CHOOSING AN ANTIBODY DETECTION METHOD IMPORTANT Read this section before beginning your experiment To detect interactions between your protein of interest and the TF proteins immobilized on the membrane you will need to use either an antibody against your protein of interest or a tag antibody such as HA biotin or GST As a negative control incubate the TranSignal TF Protein Array membrane with antibody only see Appendix B 2 1 Using an antibody against your protein of interest If you already have access to your purified protein of interest or bacterial lysate containing the pro
3. trademarks of Panomics Inc FluorChem is a registered trademark of Alpha Innotech Corporations Hyperfilm is a trademark of Amersham Pharmacia Corporation Certain aspects of the TranSignal array technology are in the process of patent filing This product is intended for research purposes only Panomics products may not be resold modified for resale or used to manufacture commercial products without written approval by Panomics Inc Panomics Inc warrants that the performance of this kit meets Panomics performance specifications from the time of shipment until the expiration date if stored under the recommended conditions Panomics disclaims all other warranties either express or implied including without limitation and implied merchantability or fitness for a particular purpose Under no circumstances shall Panomics be liable for any damages arising out of the use of the materials 2001 2005 C Panomics Inc All rights reserved MA3020UM061603 For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal TF Protein Array 1 INTRODUCTION The human genome comprises an estimated 30 000 genes 2 000 of which encode proteins involved in the control of gene expression These proteins known as transcription factors TFs are responsible for the who what when where and how of gene expression which genes are expressed in what cell types under what environmental cond
4. Place the tray on a shaker and incubate for 2 hr at room temperature Dilute the bacterial extract containing overexpressed protein to a final concentration of 60 ug ml in 4 ml of 1X Blocking Buffer I If you have purified protein dilute 3 5 ug purified protein in 4 ml of 1X Blocking Buffer I Incubate the membrane with the diluted bacterial extract or purified protein at room temperature for 2 hr with gentle shaking Wash the membrane twice with 4 ml of 1X Wash Buffer for 5 min each wash at room temperature Incubate the membrane with 4 ml of 1X Blocking Buffer II containing primary antibody Note Use an amount of the antibody suitable for western blot detection of the protein or protein tag specified by the manufacturer Incubate the membrane with the primary antibody solution for 2 hours at room temperature Wash the membrane twice with 4ml 1X Wash Buffer for 5 min each wash Incubate the membrane with the appropriate secondary antibody diluted 1 5 000 1 15 000 in 4ml 1X Blocking Buffer II for 1 hr at room temperature Wash the membrane four times with 4 ml of 1X Wash Buffer for 5 min each wash at room temperature For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal TF Protein Array 6 DETECTION Important Do not let the membrane dry out during detection 6 1 Prepare 500ul of detection solution by combining 250ul Detection Buffer A and 250 ul of Detec
5. Region 1 to 465 alone Figure A and both biotinylated and unlabeled PEPCK promoter Figure B Images were acquired using FluorChem imager Alpha Innotech Specific Protein DNA interactions boxes were competed out with unlabeled probe whereas non specific binding of the probe was not For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 20 TranSignal TF Protein Array NOTES For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com
6. These spots are Schematic diagram of the TranSignal TF Protein Array The proteins on the array are spotted in duplicate HRP marker has intended for alignment Note that the notch is at the top right hand corner For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 18 TranSignal TF Protein Array C Negative Control A s B ee C ee D t E TILES EEZETTETTITTEILIT B5 12 3456 7 8 9101112131415 16 17 18 19 20 21 22 23 24 Typical results obtained with the TranSignal TF Protein Array p53 expressing bacterial lysate A p53 purified protein B and buffer only C were incubated with the TranSignal TF Protein Array membrane for 2 hours and detected by p53 monoclonal antibody Upstate Biotechnology and anti mouse Ig HRP conjugate Amersham Images were acquired using FluorChem imager Alpha Innotech As reported in the literature ATF3 position A11 A12 interacts with p53 3 For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal TF Protein Array 19 APPENDIX F Typical Results of the TranSignal TF Protein Array when screening protein DNA interactions A Typical results obtained with the TranSignal TF Protein Array IV The TF protein array IV was incubated with the biotinylated PCR product of the rat PEPCK Promoter
7. TranSignal TF Protein Array Cat MA3501 MA3508 Product User Manual Released 03 22 05 Revised 08 11 05 2 Pa nomics Transcending boundaries Panomics Inc 2003 East Bayshore Road Redwood City CA 94063 USA Tel 650 216 9736 or 877 726 6642 PANOMIC e Fax 650 216 9790 www panomics com 2 TranSignal TF Protein Array Contents 1 INTRODUCTION 2 CHOOSING AN ANTIBODY DETECTION METHOD a Se MATERIALS PROVIDED icsesiat csssate tasceessiadecossares n 4 ADDITIONAL MATERIALS REQUIRED 5 INCUBATION OF PROTEIN WITH THE ARRAY MEMBRANE x b DETECTION necrose 7 INCUBATION OF DNA WITH THE ARRAY MEMBRANE 8 TROUBLESHOOTING 9 REFERENCES APPENDIX A Schematic Diagram of the TranSignal TF Protein Array VERSION I 13 APPENDIX B Schematic Diagram of the TranSignal TF Protein Array VERSION l 14 APPENDIX C Schematic Diagram of the TranSignal TF Protein Array VERSION Ill 15 APPENDIX D Schematic Diagram of the TranSignal TF Protein Array VERSION Iv 16 APPENDIX E Typical Results of the TranSignal TF Protein Array when screening protein protein intercictions siiis asis 17 APPENDIX E Typical Results of the TranSignal TF Protein Array when screening DNA protein interactions esssssssscscscscssscsssssesssesssssssuussssssssssssssseeessssseeseseeeeceeseseseseeeees 19 Trademarks Patents and Limited Warranty Panomics and TranSignal are
8. dy is too low Volume of detection buffer is too low Air bubbles on membrane surface during detection 11 Recommendation Check protein concentration by running the sample on SDS PAGE Check construct by DNA sequencing Make sure the DNA insert is in the right frame and that the protein expresses properly Protein binding may be hindered by a partially hidden tag Try using a higher concentration 5 10X of the bacterial lysate or longer binding time Further dilute bacterial lysate or use purified protein Further dilute the antibody Dilute the Detection Buffer Keep the membrane fully submerged in solution during all incubation steps Increase the volume to make sure that the membrane is fully submerged during incubation Increase the volume to make sure that the membrane surface is fully covered Remove air bubbles from membrane surface For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 12 TranSignal TF Protein Array TranSignal TF Protein Array 13 9 REFERENCES APPENDIX A Schematic diagram of the 1 Sawyer TK 2001 Decifering therapeutic targets BioTechniques 30 1086 TranSignal TF Protein Array Version 1090 4 vd S58 2 Chan H M and La Thangue N B 2001 p300 CBP protiens HAT for _ _ ss trancriptional bridges and
9. itions and at what stages of development The activity of TFs is highly regulated by various mechanisms including protein induction modification translocation degradation and inhibition 1 Before activated TFs can impose regulation on the basal transcriptional machinery they either bind to their corresponding specific cis elements or interact with other TF proteins This interaction usually requires cofactors to bridge the TF proteins and the components of the basal transcriptional machinery The interactions between TFs cofactors and basal transcriptional machinery create a complex multidimensional network 2 Identifying and characterizing the interaction network in the context of different cellular environments will allow us to understand the network structure as well as the cellular signal induced changes in this structure Tools for studying protein protein interactions especially interactions involving TF proteins are essential for understanding gene expression and regulation Novel method for investigating protein protein interactions Traditionally protein protein interactions have been studied by pull down assay coimunoprecipitation super gel shift and the yeast two hybrid system But because these methods are notoriously time consuming and inefficient they are not conducive to mapping the intricate network of protein protein interaction That s where the TranSignal TF Protein Array comes in This method enab
10. les you to determine how a particular protein interacts with multiple other proteins in a single detection experiment Figure 1 illustrates how this simple procedure works The array membrane is spotted with transcription factor proteins which are expressed from full length TF cDNAs with an N terminal His Tag A protein of interest is For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 3 4 TranSignal TF Protein Array used as bait to search for interactions with the immobilized proteins Interactions can be assessed either by using an antibody to the protein of interest or with antibody to protein tags such as HA GST or biotin The signal is visualized via chemiluminescent detection Insert gene of interest into expression vector HO O express your protein of interest prepare bacterial extract Sa purify protein use extract directly incubate with TranSignal TF Protein Array chemiluminescent detection Signal strength corresponds to strength of protein protein interaction Figure 1 Flow chart of the TranSignal TF Protein Array protein interaction For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal TF Protein Array Novel method to characterize gene promoter regions The TranSignal TF Protein Array provides a method by which you can very quickly survey direct interactions between TF
11. nal TF Protein Array 2 each e 4 well plate 1 each e 5X Binding Buffer 5 ml e 20X Wash Buffer 20 ml dilute to 1X with dH 0 e Streptavidin HRP 20 ul e Detection Buffer A 600 ul e Detection Buffer B 600 ul Sufficient quantities of each buffer are provided for two assays 4 ADDITIONAL MATERIALS REQUIRED 4 1 Reagents and Solutions e Appropriate primary detection antibody e Anti rabbit mouse or goat HRP conjugate 4 2 Materials and Equipment e Microcentrifuge e Orbital shaker e Plastic Sheets e g overhead transparencies e Hyperfilm ECL Amersham Cat RPN1674K or equivalent OR e Chemiluminescence imaging system e g FluorChem from Alpha Innotech Corp For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 7 8 TranSignal TF Protein Array 5 INCUBATION OF PROTEIN WITH THE ARRAY MEMBRANE Use this protocol with catalogue items MA3501 MA3502 MA3503 and MA3504 This section describes incubating bacterial extract containing your protein of interest or purified protein with the array membrane Note that the array membranes are notched at the top right hand corner for orientation purposes Note Be sure that the membrane is fully submerged in assay buffer at all times Never let the membrane dry out 5 1 5 2 5 3 5 5 5 6 5 7 5 8 59 Place each membrane into a container containing 4 ml of 1X Blocking Buffer I
12. s assay Note Be sure that the membrane is fully submerged in assay buffer at all times Never let the membrane dry out 7A 7 2 7 3 7 5 7 6 7 7 7 8 7 9 Place each membrane into a container containing 4 ml of 1X Binding Buffer Place the tray on a shaker and incubate for 2 hr at room temperature Dilute 50 200 ng of biotinylated DNA probe in 4 ml of 1X Binding Buffer For the competition assay cold probes should be in at least 5 X excess of the biotinylated probe 0 25 1 ug Incubate the membrane with the diluted DNA probe at room temperature for 30 min with gentle shaking Wash the membrane two times with 4 ml of 1X Wash Buffer for 3 min each wash at room temperature Dilute the Streptavidin HRP 1 1000 fold in 1X Wash Buffer and incubate the membraneg with the diluted Streptavidin HRP for 30 min at roomtemperature Wash three times with 4 ml of 1X Wash Buffer for 5 min each wash at room temperature Continue with step 6 1 for detection For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal TF Protein Array 8 TROUBLESHOOTING GUIDE Problem Weak or no signal High background Uneven background Not enough protein Tag is partially hidden Concentration of bacterial lysate is too high Antibody concentration is too high Membrane dried out during incubation Volume of blocking solution bacterial lysate or antibo
13. scaffolds J Cell Science 114 2363 2373 T ai 548 lt E p T i yx 3 Chunhong Yan Heng Wang and Douglas D Boyd 2002 ATF3 Represses ae L 5 72 kDa Type IV Collagenase MMP 2 Expression by Antagonizing p53 as s dependent trans Activation of the Collagenase Promoter J Bio Chem 277 Ss 10804 10812 S S sS v E 32 tS RS L sF z2 ss va gt 8 aus RER RSs S ae wes TE B35 x 2 M on SEE aa a Bi gis DLE en DV i Bes SYE KANES gs P Ses SSE For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal TF Protein Array 15 14 TranSignal TF Protein Array Schematic diagram of the TranSignal TF Protein Array Version III APPENDIX C ic diagram of the Schemat TranSignal TF Protein Array Version Il APPENDIX B uausod pupy 1ysl4 dol ay 1D S1 YJOU AY JDY AION Juawusypd 4of papuaqur ap sjods asay aupiquiau ayy fo pz gz uunjos apis 14811 ay Suojp ayvaydnp ul puv q MOA wonoq ay Buoj payjods uaaq spy AayADW JJH aiwaydnp u1 payods ain Ap tiv ayy uo sujajoid ay Apsdy U19404d AL DUSIG UDALL ay fo wyaAsvip JyDMayIS TAL HL CY W 48 T E AK 6 sada Hn AG wi a
14. tein of interest you can get started right away Simply incubate the array membrane with your purified protein then detect using the antibody 2 2 Using a protein tag antibody combination If you don t have an antibody against your protein of interest you will need to use protein with a specific tag such as HA or GST or biotinylate your protein Do not use His tagged proteins for hybridization with the membranes as the proteins on the array are His tagged You can then detect hybridized proteins using an antibody against the tag 2 3 Using a protein tag antibody combination We provide a suitable streptavidin HRP conjugate for use with this kit 3 MATERIALS PROVIDED For Catalogue items MA3501 MA3502 MA3503 and MA3504 Protein Protein interactions STORAGE CONDITONS Upon receipt store array membranes and all reagents at 4 C Keep at 4 C until use e TranSignal TF Protein Array 2 each e 1X Blocking Buffer I 30 ml e 1X Blocking Buffer II 30 ml e 20X Wash Buffer 20 ml dilute to 1X with dH 0 For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com TranSignal TF Protein Array e Detection Buffer A 600 ul e Detection Buffer B 600 ul For Catalogue items MA3505 MA3506 MA3507 and MA3508 DNA Protein interactions STORAGE CONDITONS Upon receipt remove the 5X Blocking Buffer and store at 20 C Store array membranes and all other reagents at 4 C e TranSig
15. tion Buffer B per membrane 6 2 Using forceps to hold the notched corner carefully remove each membrane from its tray Drain the excess Wash Buffer from the membrane by touching the edge against tissue Place membrane protein side up on a clean plastic sheet protector or overhead transparency by orienting the notch to the top right hand corner 6 3 Pipet the mixed Detection Buffers onto the membrane Overlay the membrane with a second plastic sheet and ensure that the buffer mixture is evenly distributed over the membrane without air bubbles 6 4 Incubate for 5 min at room temperature 6 5 Remove excess substrate by gently applying pressure to the top sheet Using a paper towel remove excess detection solution remaining on the surface of the sheets 6 6 Expose the membranes using either Hyperfilm ECL or a chemiluminescence imaging system such as the FluorChem imager from Alpha Innotech Corp In either case we recommend that you try several different exposures of varying lengths of time e g 30 sec 5 min For Technical Support call 1 877 726 6642 PANOMIC or visit our Web site at www panomics com 10 TranSignal TF Protein Array 7 INCUBATION OF DNA WITH THE ARRAY MEMBRANE Follow this protocol for use with catalogue items MA3505 MA3506 MA3507 and MA3508 This section describes incubating a biotinylated oligo of interest with the array membrane We recommend that you use a 5 biotinylated Oligo for thi
Download Pdf Manuals
Related Search