TranSignal™ SH3 Domain Array - The Jena Protein
Contents
1. pEXP Vector Ligand expression vector 10 ul 0 08 ug l 20X Wash Buffer 2 x 60 ml 1X Resuspension Buffer 60 ml ADDITIONAL MATERIALS REQUIRED Reagents and Solutions DH5a Competent Cells Gibco BRL Cat No 18265 017 Penta His Antibody BSA free Qiagen Cat No 34660 Anti Mouse IgG Fab specific Peroxidase Sigma Cat No A 2304 SuperSignal Elisa Pico Chemiluminescent Substrate Pierce Cat No 37070 SuperBlock Blocking Buffer Pierce Cat No 37545 LB w Amp Teknova Cat No 0181 A100 1 0 Tryptone 0 5 yeast extract 1 0 NaCl with 100 ug ml ampicillin IPTG Teknova Cat No 13307 50 Materials and Equipment Microcentrifuge Sonicator Small plastic tray or containers Shaker Hyperfilm ECL Amersham Cat RPN3114K or equivalent OR Chemiluminescence imaging system e g FluorChem from Alpha Innotech Corp For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com TRANSIGNAL SH3 DOMAIN ARRAY 4 PREPARE BACTERIAL EXPRESSION CONSTRUCT Using the Ligand Expression Vector provided insert the SH3 ligand of interest using standard molecular cloning techniques 6 For vector map see Appendix B Transform DH5a competent cells with DNA ligation mix as described by the manufacturer s instructions 5 PREPARE BACTERIAL EXTRACTS WITH SH3 LIGAND In this section you will prepare bacterial extract containing your ligand of interest to hybridize with the arra2. TranSignal SH3 Domain Array Catalog No MA3010 Product User Manual Version 1 1 E a LEE Pa no m ICS Transcending boundaries Panomics Inc 2003 East Bayshore Road Redwood City CA 94063 USA Tel 650 216 9736 or 877 726 6642 PANO MIC Fax 650 216 9790 www panomics com 2 TRANSIGNAL SH3 DOMAIN ARRAY CONTENTS T INTRODUCTION sc astitit ett o tastes Te tonal detested alae antem 3 2 MATERIALS PROVIDED sott tta bte a rete p etd p te 6 3 ADDITIONAL MATERIALS REQUIRED sisirin 6 4 PREPARE BACTERIAL EXPRESSION CONSTRUCT nn 7 5 PREPARE BACTERIAL EXTRACTS WITH SH3 LIGAND enne 7 Bie INCUBATION ccc oi te Rt ED ode De deed qu ies 8 To WASH Ge DETECT niinniin hoped nda 8 Bs TROUBIESHOOTING costes editt mper ta deb tematicas 9 Ds REFERENCES iiin anina aina 10 APPENDIX A TYPICAL RESULTS OF TRANSIGNAL SH3 DOMAIN ARRAY 11 APPENDIX B SCHEMATIC DIAGRAM OF THE TRANSIGNAL SH3 DOMAIN ARRAY 11 APPENDIX C MAP amp MCS OF EXPRESSION VECTOR enn 13 Trademarks Patents and Limited Warranty Panomics is a trademark of Panomics Inc SuperSignal is a trademark of Pierce FluorChem is a trademark of Alpha Innotech Corporation 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 purpose only Panomics products may not b
3. aining your SH3 ligand of interest Sequence Information for this vector can be downloaded from our web site at www panomics com For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com 14 TRANSIGNAL SH3 DOMAIN ARRAY NOTES For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com TRANSIGNAL SH3 DOMAIN ARRAY 15 NOTES For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com
4. associated binding protein Mixed lineage protein kinase 3 Nebulin Bruton Tyrosine Kinase Intersectin SH3 Domain 2 Yamaguchi sarcoma virus oncogene homolog 1 Cellular Rous Sarcoma Virus GTPase activating protein Intersectin SH3 Domain 1 Lyn protein non receptor kinase Fyn binding protein Presynaptic density protein 95 TXK tyrosine kinase Spectrin alpha chain Hemopoietic cell kinase Rho guanine nucleotide exchange factor GEF 5 Glutathione S transferase Interleukin 2 inducible T cell kinase Vav 2 oncogene product SH3 Domain 2 Hematopoietic specific protein 1 Avian sarcoma virus CT10 oncogene homolog SH3 Domain 2 Neurite outgrowth factor Abl SH3 Domain 2 For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com TRANSIGNAL SH3 DOMAIN ARRAY 13 APPENDIX C MAP amp MCS OF EXPRESSION VECTOR Histag tetramer polyG MCS 406 483 P pEXP VECTOR 2 7 kb 790 Amps Multiple Cloning Site MCS 406 483 bp mu GCA TGC CTG CAG GTC GAC TCT AGA GGA TCC CCG GGT ACC GGT ACT AGT AGA AM AAT GAG TAA CGG CCG TAC GGG CCC Sph Pst Xba Xmal Koni Agel Eag Apa Sma Spe BsiW Description The ligand expression vector is designed for use with the TranSignal SH3 Domain Array Insert your SH3 ligand of interest using standard molecular cloning techniques Then follow the protocol as described in Section 5 to prepare bacterial extracts cont
5. e TranSignal SH3 Domain Array you can determine whether your protein of interest binds to one or more of the 38 different SH3 domains all in one experiment The assay couldn t be simpler just express your protein of interest in bacteria and incubate the extract with the TranSignal SH3 Domain Array membrane The protein interactions literally take place on the array membrane and you can visualize them using chemiluminescent detection We ve spotted 38 of the most commonly studied SH3 domains on the TranSignal SH3 Domain Array For a complete map of the array and list of SH3 domains see the Appendix For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com TRANSIGNAL SH3 DOMAIN ARRAY Insert SH3 ligand of interest using ligand expression vector Q0 done amp express your protein of interest extrad incubate extract or protein with TranSignal SH3 Domain Array membrane prepare bacterial chemiluminescence detection Signal strength corresponds to strength of protein protein interaction Figure 1 Flow chart of the TranSignal SH3 Domain Array procedure For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com 5 6 3 3 1 3 2 TRANSIGNAL SH3 DOMAIN ARRAY MATERIALS PROVIDED TranSignal SH3 Domain Array 2 each Positive Control Bacterial Extract 1 5 ml From E Coli containing Class Ib Ligand from P13K
6. e 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 meet 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 2 Panomics Inc All rights reserved UM22302129 For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com TRANSIGNAL SH3 DOMAIN ARRAY 1 INTRODUCTION One of the keys to understanding cellular signal transduction is clarifying how proteins interact with one another Protein protein interactions are often mediated by noncatalytic conserved domains One of these domains is the Src homology 3 or SH3 domain 1 SH3 Domain Structure amp Function First identified as part of the Rous sarcoma oncogene product src SH3 src homology 3 domains play an important role in intercellular communication and intracellular signal transduction Each SH3 domain is a small conserved sequence of about 60 amino acids that interacts with proline rich binding sites These sites known as SH3 ligands contain 6 to 12 residues with a con
7. gged ligand has been spotted along the right and bottom sides of the membrane These spots are 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 website at www panomics com 12 TRANSIGNAL SH3 DOMAIN ARRAY Table 1 List of SH3 Domains COORD Al 2 A3 4 A5 6 A7 8 B1 2 B3 4 B5 6 B7 8 C1 2 C3 4 C5 6 C7 8 D1 2 D3 4 D5 6 D7 8 E1 2 E3 4 E5 6 E7 8 F1 2 F3 4 F5 6 F7 8 G1 2 G3 4 G5 6 G7 8 H1 2 H3 4 H5 6 H7 8 I1 2 13 4 SH3 DOMAINS Amphiphysin Dlg2 VAV BLK CCB4 EMP55 NCK 3 Abl SPCN FGR Y124 PLCr Cortactin SLK PEXD Riz MLPK3 Nebulin BTK ITSN 2 Yes1 c Src RasGAP ITSN 1 Lyn FYB PSD95 TXK SJHUA Hck Tim GST Itk VAV2 2 HS1 CRK 2 NOF Abl 2 FULL NAME Amphiphysin Discs large homolog 2 Vav 1 oncogene product Beta lymphocyte specific protein tyrosine kinase Dihydropyridine sensitive L type calcium channel beta 4 subunit 55K erythrocyte membrane protein SH3 domain 3 of the melanoma cDNA encoding a cytoplasmic protein conisisting of the src homology units SH2 and SH3 Abelson tyrosine kinase SH3 Domain 1 Spectrin alpha chain non erythrocytic Cellular Gardner Rasheed feline sarcoma virus protein PAK interacting exchange factor beta Phospholipase C gamma 1 Cortactin Src like kinase Peroxisomal membrane protein pex13 Retinoblastoma
8. hidden Primary or secondary antibody is no longer working Nonspecific interaction with antibodies or other reagents used in the assay The ligand concentration is too high The blocking solution is not working properly Secondary antibody concentration is too high The volume of primary antibody is too low Recommendation Check construct by DNA sequencing Ensure that the cloned insert does not contain an internal translational start site Protein binding may be hindered by a partially hidden His tag Try using a high concentration 5 10X of the bacterial lysate or use longer binding time Check by dot blot to determine if antibodies are working properly Check signal using a zero standard i e PVDF membrane alone High background is usually the result of the antibody system used Try using a less sensitive substrate such as ECL Dilute the bacterial lysate 30X Test the blocking solution with western blot or positive control membrane Lower the concentration of secondary antibody Increase the volume to make sure that the membrane is fully covered during incubation For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com 10 TRANSIGNAL SH3 DOMAIN ARRAY if REFERENCES Pawson T 1995 Protein modules and signalling networks Nature 373 573 580 Lowenstein E J Daly R J Batzer A G Li W Margolis B Lammers R Ull
9. ns Place the tray on shaker and incubate for 30 min at room temperature Remove 1X SuperBlock Blocking Buffer and briefly rinse membrane two times with 1X Wash Buffer Incubate with 30 ml of diluted bacterial extract from Step 5 12 or use the Positive Control Extract provided at room temperature for 1 hr or 4 C overnight WASH amp DETECT After incubation wash three times with 40 ml of 1X Wash Buffer for 5 min each wash Incubate with Penta His Antibody 1 2000 dilution in 1X Wash Buffer for 1 hr at room temperature Wash three times with 40 ml of 1X Wash Buffer for 5 min each wash Incubate with Anti mouse IgG Peroxidase 1 2000 dilution in 1X Wash Buffer for 1 hr at room temperature Wash three times with 40 ml of 1X Wash Buffer for 5 min each wash Detect using SuperSignal Substrate from Pierce We recommend 1 ml of working solution per membrane 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 website at www panomics com TRANSIGNAL SH3 DOMAIN ARRAY 8 TROUBLESHOOTING 9 Problem Weak or no signal High background Uneven background Cause Expressed ligand does not have a His tag His tag is partially
10. our website at www panomics com TRANSIGNAL SH3 DOMAIN ARRAY 11 APPENDIX A TYPICAL RESULTS OF TRANSIGNAL SH3 DOMAIN ARRAY A B 1 2 12345678 b a 3 a 4 30 kD a 5 6 g 8 15 kD ABCDEFGHIJ Figure 1 Typical results of TranSignal SH3 Domain Array Panel A Bacterially expressed class Ib SH3 ligand specifically interacts with corresponding SH3 domains Class Ib encoding sequence was inserted into the Ligand Expression Vector and DH5a was transformed with the resulting construct Bacterial extract from the transformed cells was hybridized with the TranSignal SH3 Domain Array and the image was acquired using FluorChem imager from Alpha Innotech Spots with higher intensity indicate higher binding affinity with ligand of interest to SH3 Domain s Panel B Class Ib SH3 ligand specifically pulls down its corresponding SH3 Domains Lane 1 marker Lane 2 ligand Lane 3 GST Lane 4 EMP55 Lane 5 CCB4 Lane 6 c Src Lane 7 Lyn Lane 8 Yes APPENDIX B SCHEMATIC DIAGRAM OF THE TRANSIGNAL SH3 DOMAIN ARRAY Cortactin Yesl 1 Cortactin Yesl 2 EMP55 Nebulin cSrc NOF 3 EMP55 Nebulin c Src NOF 4 NCK 3 BIK RasGAP Abl 2 5 NCK 3 BIK RasGAP Abl 2 6 Abl 1 ITSN2 ITSN 1 1 A B C D E F G H J Figure 2 Schematic diagram of the TranSignal SH3 Domain Array The proteins on the array are spotted in duplicate the first row is protein spotted normally the second row is diluted 1 5 His ta
11. rich A Skolnik E Y Bar Sagi D and Schlessinger J 1992 The SH2 and SH3 domain containing protein GRB2 links receptor tyrosine kinases to ras signaling Cell 70 431 42 Bar Sagi D Rotin D Batzer A Mandiyan V and Schlessinger J 1993 SH3 domains direct cellular localization of signaling molecules Cell 74 83 91 Gout I Dhand R Hiles I D Fry M J Panayotou G Das P Truong O Totty N F Hsuan J Booker G W et al 1993 The GTPase dynamin binds to and is activated by a subset of SH3 domains Cell 75 25 36 Pleiman CM Hertz WM Cambier JC 1994 Activation of phosphatidylinositol 3 kinase by Src family kinase SH3 binding to the p85 subunit Science 263 1609 1612 Alexandropoulos K Cheng G and Baltimore D 1995 Proline rich sequences that bind to Src homology 3 domains with individual specificities Proc Natl Acad Sci USA 92 3110 3114 Rickels R J Botfield M C Zhou X M Henry P A Brugge J S and Zoller M J 1995 Phage display selection of ligand residues important for Src homology 3 domain binding specificity Proc Natl Acad Sci USA 92 10909 13 Weng Z Rickles RJ Feng S Richard S Shaw A S Schreiber S L and Burgge J S 1995 Structure function analysis of SH3 domains SH3 binding specificity altered by single amino acid substitutions Mol Cell Bio 15 56327 34 For Technical Support call 1 877 726 6642 PANOMIC or visit
12. served Pro Xaa Xaa Pro PXXP motif 1 SH3 domains act as part of an adapter molecule recruiting downstream proteins in a signaling pathway SH3 domains mediate interactions in many key signaling pathways including epidermal growth factor receptor signaling 2 cellular localization of cytoplasmic proteins 3 upregulation of GTPase activity of dynamin 4 and activation of phosphatidylinositol 3 kinase in response to IgM crosslinking 5 And SH3 domain activity has been implicated in both cancer and AIDS Studying SH3 Domains As we near completion of the human genome project new protein identities will emerge as will a growing need to study their functions One way to characterize protein function is to identify which SH3 domain it binds to and hence unlock which signaling pathway it is involved with In order to dissect signaling pathways that involve SH3 domains scientists need a way to determine whether their ligand of interest interacts with SH3 domains and specifically which SH3 domains bind to it But this requires an assay that allows the SH3 domain to stay folded in its active conformation Traditional methods for assaying protein protein interactions such as co immunoprecipitation are arduous and time consuming at best For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com 3 4 TRANSIGNAL SH3 DOMAIN ARRAY Observe protein interactions using all in one array system With th
13. y membrane Section 6 5 1 Inoculate the transformed bacteria in LB Amp 100 ug ml Section 4 5 2 Grow bacteria overnight at 37 C with shaking 225 rpm 53 Transfer 40 ul of overnight culture to a tube containing 4 ml of LB Amp 100 ug ml 5 4 Grow bacteria at 37 C until OD600 readings are approx 0 5 0 8 55 Add 100 uM IPTG 5 6 Continue to grow for an additional 1 5 2 5 hr 5 7 Centrifuge cells at 4 000 rpm for 5 min Decant supernatant 5 8 Resuspend pellet in 750 ul of ice cold 1X Resuspension Buffer provided 5 9 Lyse cells using a sonicator 5 10 Centrifuge at 14 000 rpm for 1 min at 4 C 5 11 Transfer supernatant into a clean microcentrifuge tube 5 12 Dilute bacterial extract to a final concentration of 0 1 ug ul in 1X Resuspension Buffer 5 13 Store on ice until further use For longer storage keep at 20 C For Technical Support call 1 877 726 6642 PANOMIC or visit our website at www panomics com 8 6 TRANSIGNAL SH3 DOMAIN ARRAY INCUBATION In this Section you will incubate the bacterial extract containing your SH3 ligand of interest prepared in Section 5 to the array membrane Be sure to prepare additional reagents not included with this kit as described by the manufacturer 6 1 6 2 6 3 6 4 7 7 1 7 2 7 3 7 4 7 5 7 6 Place each membrane into a small tray containing 30 ml of 1X SuperBlock Blocking Buffer prepare buffer according to manufacturer s instructio
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