50 nmoles substrate. 8,000 Test Points based on 100 nm in reaction

Transcription

1 Product Insert IMAP Substrates About the IMAP Substrates To facilitate the customization of the IMAP assay to fit your specific needs, Molecular Devices offers a wide range of validated substrates and calibrators. The IMAP substrates, which have been optimized for maximum performance in FP and TR-FRET detection, are offered individually as a complement to the IMAP Screening Express and IMAP Purchase Plan (IPP). The IMAP Screening Express and IPP programs allow greater flexibility by providing the detection portion of the IMAP assay customized for each substrate sequence, leaving the enzymatic reaction to be determined by the researcher. These substrates may be used for the enzymes for which they were tested or as potential substrates for alternate enzymes. Table 1 lists the substrates, their amino acid sequences, part numbers, recommended Binding Solution formulation and binding incubation times 1 for FP and TR-FRET detection. Due to the greater flexibility in substrate concentration using TR-FRET detection, the substrate amounts are given now as nmoles/vial rather than dp/vial. Table 2 lists the IMAP PDE substrates, their corresponding conditions and their part numbers. For more information and data about subtsrates and enzymes assayed with the IMAP system please visit our IMAP Assay Archive under: Table 1. IMAP Peptide Substrates and Calibrators Quantity: 8,000 (50 nmoles) or 50,000 (312.5 nmoles) test points based on 100 nm end concentration in kinase reaction 2 Product Name Peptide Sequence 3 Enzymes assayed with the substrate 4 50 nmoles substrate 8,000 Test Points based on 100 nm in reaction nmoles substrate IMAP Progressive Binding System 1,5 Original Binding System 6 Detection 50,000 Test System (TR- Points based on FRET Tb Binding Minimum 100 nm in donor: 1/400 1x Buffer 1x Buffer Reagent Binding Binding Reagent reaction for all) A (%) B (%) Dilution Incubation Time Dilution FAM-Abltide 5FAM-KKGEAIYAAPFA-NH 2 Abl, Arg R7253 R7258 FP /600 1 hour 1/400 TAMRA-Abltide 5TAMRA-KKGEAIYAAPFA-NH 2 Abl, Arg R7374 R7375 FP /600 1 hour Not Recommended FAM-acidic α tubulin derived 5FAM-ALEKDYEEVGV-NH2 Syk RP7104 RP7604 FP /1500 overnight Not tested buffer 5 TR-FRET /1000 overnight Not tested FAM-ADR1- derived 5FAM-LKKLTRRASFSGQ-COOH CaM KII RP7004 RP7504 FP / minutes Not tested FAM-Alternate Syntide2 5FAM-PLSRTLSVSSLPGL-NH 2 c-tak RP7045 RP7545 FP / minutes Not tested TR-FRET /400 1 hour Not tested FAM-AMPKtide 5FAM-LKKLTRRPSFSAQ-COOH AMPK RP7001 RP7501 FP / minutes Not tested IMAP Peptide Substrate 1

2 FAM-Autocamtide2 5FAM-KKALRRQETVDAL-NH 2 CaM KII RP7041 RP7541 FP /600 1 hour Not tested TR-FRET /800 overnight Not tested FAM-Autocamtide3 5FAM-KKALHRQETVDAL-NH 2 CaM KII R7368 R7369 FP /600 1 hour Not Recommended TR-FRET /800 overnight Not tested TAMRA- Autocamtide3 5TAMRA-KKALHRQETVDAL-NH 2 CaM KII R7333 R7334 FP /600 1 hour Not Recommended TR-FRET /600 overnight Not tested Blk, Lyn, Bmx, Flt3, FAM-Blk/Lyntide 5FAM-EFPIYDFLPAKKK-NH 2 ITK, Lck TrkA, TIE2 R7188 R7233 FP /1200 2h 1/400 TR-FRET /800 overnight Not tested TAMRA-Blk/Lyntide 5TAMRA-EFPIYDFLPAKKK-NH 2 Blk, Lyn R7358 R7359 FP /600 1 hour 1/400 TR-FRET /600 overnight Not tested FAM-Phospho Blk/Lyntide 5FAM-EFPI-pY-DFLPAKKK-NH 2 Calibrator R7354 R7355 FP /600 1 hour 1/400 TR-FRET /800 overnight Not tested FAM-BTK derived Axl, EphB2, EphB3, 5FAM-KKVVALYDYMPMN-NH2 JAK3, BTK RP7053 RP7553 FP / minutes Not tested buffer 5 FAM-Cdc25C-derived 5FAM- VSRSGLYRSPSMPENLNRPR- COOH PRK2, PAR1Ba R7275 R7276 FP /600 1 hour 1/400 FAM-CDK7 derived 5FAM-FLAKSFGSPNRAYKK-OH Nek6 RP7141 RP7641 FP / minutes Not tested buffer 5 TR-FRET /600 4h Not tested TAMRA-CDK7tide 5TAMRA- YSPTSPSYSPTSPSYSPTSPS- CDK7 R7352 R7353 FP / minutes 1/400 COOH FAM-CHK1tide 5FAM-ALKLVRYPSFVITAK- NH 2 CHK1 R7185 R7230 FP / minutes 1/200 TR-FRET /600 2 hours Not tested TAMRA-CHK1tide 5TAMRA-ALKLVRYPSFVITAK- COOH CHK1 R7343 R7344 FP / minutes Not Recommended FAM-CHK2tide 5FAM-AMRLERQDSIFYPK- NH 2 CHK2 R7186 R7231 FP /600 1 hour 1/400 TR-FRET /600 overnight Not tested TAMRA-CHK2tide 5TAMRA-AMRLERQDSIFYPK- NH 2 CHK2 R7360 R7361 FP /600 1 hour 1/400 TR-FRET /600 overnight Not tested FAM-CK1tide 5FAM-HAAIGDDDDAYSITA- NH 2 CK1, CK1δ R7311 R7312 FP /1500 overnight Not Recommended 0.1% BSA rxn buffer 5 TR-FRET Not tested Not tested IMAP Peptide Substrate 2

3 FAM-CK1tide 5FAM-HAAIGDDDDAYSITA- NH 2 CK1, CK1δ R7311 R7312 FP and 0.03% BSA 7 buffer 5 TR-FRET /1000 overnight Not tested TAMRA-CK1tide 5TAMRA-HAAIGDDDDAYSITA-NH 2 CK1, CK1δ R7370 R7371 FP / hours Not Recommended TR-FRET Not recommended Not tested FAM-CK2tide 5FAM-RRRADDSDDDDD-NH 2 CK2α R7294 R7295 FP and 9% 5M NaCl 1:2500 overnight Not Recommended TR-FRET /800 overnight Not tested FAM-phospho-CK2tide 5FAM-RRRADD-pS-DDDDD-NH 2 Calibrator R7347 R7348 FP and 9% 5M NaCl overnight Not Recommended TR-FRET /800 overnight Not tested FAM-CREBtide 5FAM-GEILSRRPSYRK-NH 2 MSK1 RP7046 RP7546 FP / minutes Not tested TR-FRET /400 1 hour Not tested FAM-Crosstide 5FAM-GRPRTSSFAEG-COOH Akt1, Akt2, Akt3, MSK1, MSK2, SGK1, R7110 R7136 FP /600 1 hour 1/400 Plk3 TR-FRET /800 overnight Not tested TAMRA-Crosstide 5TAMRA-GRPRTSSFAEG-COOH Akt1, Akt2, Akt3, MSK1, MSK2, SGK1 R7305 R7306 FP /600 1 hour 1/400 TR-FRET /1000 overnight Not tested FAM-phospho-Crosstide 5FAM-GRPRTS-pS-FAEG-COOH PP2A, PP1; Calibrator R7159 R7171 FP /600 1 hour 1/600 TR-FRET /800 overnight Not tested TAMRA-phospho- 5TAMRA-GRPRTS-pS-FAEG - Crosstide COOH PP2A, PP1;Calibrator R7321 R7322 FP /600 1 hour 1/600 TR-FRET /1000 overnight Not tested FAM-CSKtide 5FAM-KKKKEEIYFFFG-NH Axl, CSK, Fes, Flt1, 2 FGFR3, KIT, TIE2 R7269 R7270 FP / hours 1/400 TR-FRET /1000 overnight Not tested FAM-EGFR-derived TAMRA-EGFR-derived FAM-EGFR-derived phospho LVEPLTPSGEAPNQK-5FAM-COOH JNK1, JNK2, JNK3, p38 (α,β,γ,δ isoforms) R7129 R7170 FP / hour 1/400 LVEPLTPSGEAPNQK-5TAMRA-NH 2 p38 (α,β,γ,δ isoforms) R7307 R7308 FP /600 1 hour 1/400 TR-FRET /800 4 hours Not tested LVEPL-pT-PSGEAPNQK -5FAM- COOH Calibrator R7319 R7320 FP /600 1 hour 1/400 IMAP Peptide Substrate 3

4 FAM-Erktide IPTTPITTTYFFFK-5FAM-COOH Erk1, Erk2, p38 (α,β,γ,δ isoforms) R7292 R7293 FP / hours Not Recommended 0.1% BSA rxn buffer 5 TR-FRET Not tested Not tested FAM-Erktide IPTTPITTTYFFFK-5FAM-COOH Erk1, Erk2, p38 (α,β,γ,δ isoforms) R7292 R7293 FP /600 1 hour Not Recommended buffer 5 TAMRA-p38tide 5TAMRA-IPTTPITTTYFFFK-NH 2 Erk2 R7350 R7351 FP / minutes Not Recommended FAM-FcεRI γ-chain ITAM-derived phospho TAMRA- FcεRI γ-chain ITAM-derived phospho 5FAM-pY-TGLSTRNQET-pY-ETL- NH 2 PTP1B R7187 R7232 FP /600 1 hour 1/800 TR-FRET /600 overnight Not tested 5TAMRA-pY-TGLSTRNQET-pY-ETL- NH 2 PTP1B R7339 R7340 FP /600 1 hour 1/800 TR-FRET /800 4 hours Not tested FAM-Forkhead-derived 5FAM-KKISGRLSPIMTEQ- NH 2 DYRK 1A R7315 R7316 FP / minutes 1/400 FAM-Gastrin-derived 5FAM-GPWLEEEEEAYGWMDFK NH 2 PDGFR α R7366 R7367 FP /2500 overnight Not Recommended buffer 5 TR-FRET /1000 overnight Not tested FAM-Gastrin-derived 5FAM-GPWLEEEEEAYGWMDFK NH 2 PDGFR α R7366 R7367 FP /2500 overnight Not Recommended 0.1% BSA rxn buffer 5 TR-FRET Not tested Not tested FAM-Glycogen Synthase-derived TAMRA Glycogen Synthase-derived TAMRA Glycogen Synthase-derived phospho FAM-Glycogen Synthase-derived phospho 5FAM-KKLNRTLSVA-COOH 5TAMRA-KKLNRTLSVA-COOH MAPKAP K2, PRAK, PKD2, PKCμ R7127 R7169 FP / minutes 1/400 MAPKAP K2, PRAK, PKCμ R7277 R7278 FP / minutes 1/400 5TAMRA-KKLNRTL-pS-VA-COOH Calibrator R7376 R7377 FP / minutes Not Recommended 5FAM-KKLNRTLS-pS-VA-COOH Calibrator R7317 R7318 FP / minutes 1/400 IMAP Peptide Substrate 4

5 FAM-Histone H1 FAM-Histone H1- derived TAMRA-Histone H1- derived Labeled protein 5FAM-GGGPATPKKAKKL-COOH CDK5/p35, CDK5/p25, CDK4/Cyclin D1, R7439 R7440 FP /400 1 hour Not tested CDK6/D1 CDK1/Cyclin B,CDK2/Cyclin A, CDK2/CyclinE,CDK3/ CyclinE, CDK5/p25,CDK5/p35, CDK6/Cyclin D3 R7252 R7257 FP / minutes Not Recommended 5TAMRA-GGGPATPKKAKKL-COOH CDK1/Cyclin B R7384 R7385 FP / minutes Not Recommended FAM-Histone H1-5FAM-GGGPA-pT-PKKAKKL-COOH derived phospho Calibrator R7372 R7373 FP / minutes Not Recommended FAM-IκBα-derived 5FAM-GRHDSGLDSMK-NH 2 IKKβ*, IKKα R7254 R7259 FP /600 1 hour 1/400 TAMRA-IκBα-derived 5TAMRA-GRHDSGLDSMK-NH 2 IKKβ, IKKα R7341 R7342 FP /600 1 hour 1/400 TR-FRET /800 overnight Not tested FAM-IκBα-derived phospho 5FAM-GRHDSGLD-pS-MK-NH 2 Calibrator R7301 R7302 FP /600 1 hour 1/400 FAM-IP3R-derived 5FAM-GRRESLTSFG-NH 2 PKA, PKG RP7035 RP7535 FP /600 1 hour Not tested TR-FRET /600 overnight Not tested FAM-IRS1- derived TAMRA-IRS1- derived 5FAM-KKSRGDYMTMQIG- NH 2 Insulin Receptor,IGF1- R Insulin Receptor,IGF1- R R7267 R7268 FP / minutes 1/400 TR-FRET /600 4 hours Not tested 5TAMRA-KKSRGDYMTMQIG-NH 2 R7335 R7336 FP / minutes Not Recommended TR-FRET /600 4 hours Not tested TAMRA- Kemptide 5TAMRA-LRRASLG-COOH PKA, Aurora A R7331 R7332 FP / minutes Not Recommended FAM-MDCtide 5FAM-GEYAAEYADDAYAADYYAA- TrkA, TrkB, FGFR3, NH2 Fes RP7101 RP7601 FP /1500 overnight Not tested buffer 5 TR-FRET /1000 overnight Not tested FAM-MLC-derived 5FAM-KKRPQRRYSNVF-COOH PKA, PAK1, PAK2, Aurora A RP7008 RP7508 FP / minutes Not tested IMAP Peptide Substrate 5

6 FAM-Nyk-derived 5FAM-IYSGDYYR-NH2 JAK3, IGF-1R RP7082 RP7582 FP / hours Not tested buffer 5 TR-FRET /600 overnight FAM-p34cdc2-derived Src, Fyn, Lck, Yes, 5FAM-KVEKIGEGTYGVV-NH 2 Hck, Rse R7157 R7172 FP /600 1 hour 1/400 TAMRA-p34cdc2- derived 5TAMRA-KVEKIGEGTYGVVYK-NH 2 Src, Fyn, Lck, Yes R7309 R7310 FP /600 1 hour 1/400 TR-FRET /600 overnight Not tested FAM-p34 cdc2 -derived phospho 5FAM-KVEKIGEGT-pY-GVV- NH 2 Calibrator R7271 R7272 FP /600 1 hour 1/400 Erk1, Erk2, p38 FAM-p38tide 5FAM-IPTTPITTTYFFFK-NH 2 (α,β,γ,δ isoforms) R7434 R7435 FP /600 1 hour Not tested buffer 5 FGFR3, PDGFRα, FAM-PDGFRtide 5FAM-QEEEYVFIE-NH 2 PDGFRβ RP7084 RP7584 FP /1500 overnight Not tested buffer 5 TR-FRET /800 overnight Not tested FAM-PKA regulatory subunit-derived phospho 5FAM-LDVPIPGRFDRRV-pS-VAAE- NH 2 Calcineurin* R7251 R7256 FP /600 1 hour 1/400 TR-FRET /800 4 hours Not tested FAM-PKAtide 5FAM-GRTGRRNSI-NH 2 Aurora A, PKA*, PKG R7250 R7255 FP / minutes Not Recommended TAMRA-PKAtide 5TAMRA-GRTGRRNSI-COOH PKA, PKG R7313 R7314 FP / minutes Not Recommended FAM-phospho-PKAtide 5FAM-GRTGRRN-pS-I-NH 2 Calibrator R7303 R7304 FP / minutes Not Recommended TAMRAphosphoPKAtide 5TAMRA-GRTGRRN-pS-I-COOH Calibrator R7378 R7379 FP / minutes Not Recommended FAM-PKCα pseudosubstrate derived 5FAM-RFARKGSLRQKNV-COOH PKCζ RP7032 RP7532 FP / minutes Not tested TR-FRET /400 3 hours Not tested FAM-PKCε pseudosubstrate derived 5FAM-ERMRPRKRQGSVRRRV-NH 2 PKCγ, Pim1 RP7048 RP7548 FP / minutes Not tested TR-FRET /400 3 hours Not tested IMAP Peptide Substrate 6

7 TAMRA-PKCε psuedosubstrate derived 5TAMRA- ERMRPRKRQGSVRRRV -NH 2 PKCγ, Pim1 R7329 R7330 FP / minutes 1/400 FAM-extended PLMderived 5FAM-GTFRSSIRRLSTRRR-OH Nek2, IRAK4 RP7140 RP7640 FP / minutes Not tested buffer 5 TR-FRET /400 3 hours Not tested TAMRA-PLM-derived 5TAMRA-IRRLSTRRR-COOH NEK2 R7345 R7346 FP / minutes Not Recommended FAM-S6 Ribosomal Protein-derived 5FAM-AKRRRLSSLRA-COOH ROCK-II, Rsk1,Rsk2, Rsk3, Tab1 R7184 R7229 FP / minutes 1/200 TAMRA-S6 Ribosomal ROCK-II, Rsk1, Rsk2, 5TAMRA-AKRRRLSSLRA-COOH Protein-derived Rsk3 R7323 R7324 FP / minutes 1/200 TR-FRET /600 2 hours Not tested FAM- S6 Ribosomalprotein derived 5FAM-AKRRRL-pS-pS-LRA-COOH Calibrator R7356 R7357 FP / minutes 1/400 phospho FAM-RPTKtide 5FAM-AEEEYFFLF-NH EGFR, FGFR3, 2 PDGFRβ RP7063 RP7563 FP /1750 overnight Not tested buffer 5 TR-FRET /1000 overnight Not tested TAMRA-Rostide 5TAMRA-KKKSPGEYVNIEFG-NH 2 Ros, Met R7382 R7383 FP /600 1 hour Not Recommended TR-FRET /600 overnight Not tested FAM-RS domain derived 5FAM-GRSRSRSRSR-OH SPRK1, IRAK4 RP7153 RP7653 FP / minutes Not tested buffer 5 TR-FRET /600 overnight Not tested CDK7/Cyclin H, FAM-small library PAK1, PAK4, 5FAM-KKRNRRLSVA-OH derived CDK6/CycD1, Pctaire, RP7138 RP7638 FP / minutes Not tested Plk1 buffer 5 FAM-Src Family-tide 5FAM-GEEIYGEFD-NH 2 FAK, Src RP7060 RP7560 FP /1500 overnight Not tested buffer 5 TR-FRET /400 overnight Not tested EphA1, ErbB4, FAM-Srctide 5FAM-GEEPLYWSFPAKKK-NH 2 MUSK, Src, Lck RP7095 RP7595 FP / hours Not tested buffer 5 TR-FRET /800 overnight Not tested IMAP Peptide Substrate 7

8 FAM-Synapsin I-derived TAMRA-Synapsin I- derived FAM-α-Tubulin-derived TAMRA-α-Tubulinderived 5FAM-LRRRLSDANF-NH 2 AMPK, CaMK IV, PKCμ R7273 R7274 FP / minutes 1/400 TR-FRET /800 overnight Not tested 5TAMRA-LRRRLSDANF-NH 2 PKCμ R7380 R7381 FP / minutes Not Recommended 5FAM-ALQKDYENVGV-NH 2 Syk, Zap 70 R7189 R7234 FP /600 1 hour 1/400 5TAMRA-ALQKDYENVGV-NH 2 Syk R7337 R7338 FP /600 1hour Not Recommended FAM-α-Tubulin-derived phospho 5FAM-ALQKD-pY-ENVGV-NH 2 Calibrator R7299 R7300 FP /600 1 hour 1/400 sequence resulting in the most sensitive assay for enzymes with several possible substrates NOTES: 1: The Binding Buffer conditions as described in Table 1 are a starting point for IMAP assay development. They were optimized for FP detection using 100 nm substrate in IMAP reaction buffer with BSA (containing 1 mm DTT) in the presence of 100 µm ATP unless stated otherwise. For TR-FRET detection the same DTT and ATP in 0.01% Tween containing buffer was used. If alternative reaction conditions are used, a quick background check of the substrate is recommended. 2: Number of test points is based on a typical 384-well microplate assay consisting of a 20 µl kinase reaction and 60 µl of Binding Solution. Using FP detection, the most common substrate concentration is 100 nm. This parameter is far more flexible using TR-FRET detection. 3: px = phosphorylated on X residue, 5FAM = 5-carboxyfluorescein, 5TAMRA = 5-carboxytetramethylrhodamine, -NH2 = C-terminal amide, -COOH = C-terminal free acid. 4: An IMAP response of ΔmP >100 mp has been achieved with the substrate for any enzyme listed in this column. Phosphos denoted as calibrator can be used to calibrate the IMAP response for the corresponding non-phosphorylated form. Please contact your technical sales representative if you need more information about calibration of IMAP assays. 5: 1x BSA reaction buffer : 10 mm Tris-HCl, 10 mm MgCl2, 0.1% BSA, 0.05% NaN3, ph x Tween reaction buffer: 10 mm Tris-HCl, 10 mm MgCl2, 0.01% Tween-20, 0.05% NaN3, ph : Original Binding Reagent is diluted in 1x Original Binding Buffer and FP is read 30 minutes after addition of binding solution (= binding incubation time). 7: Any commercially available BSA can be used; however phosphate-free BSA of ³ 98% purity is recommended. 8: The 1x Buffer percentages are the final concentrations in the binding solution. Suggested binding incubation time (= incubation step after addition of binding solution) varies for the Progressive Binding System, depending on Progressive Buffer proportions. Assays utilizing a greater proportion of Buffer B take longer to achieve a stable signal. For substrates which require greater than 100% 1x Buffer B, a >1x stock must be used, e.g., 160% 1x Buffer B=1.6x Buffer B. These tables are provided as a reference for the name and sequence of the substrates provided with Molecular Devices Reagent Kits. These substrates can also be used with the IMAP Screening Express and the IMAP Purchase Plan (IPP) for assay development using proprietary enzymes. All substrates are guaranteed to have 90% or greater purity by HPLC prior to lyophilization. All of these substrates have been verified for use with the IMAP platform. Please refer to IMAP product literature for detailed assay protocols. Please contact your technical sales representative if there are any questions concerning the ordering of these substrates or questions regarding the Screening Express or IMAP Purchase Plan. IMAP Peptide Substrate 8

9 The following protocols are for lyophilized substrates. Substrates should be used according to the assay protocols supplied with the Screening Express, IMAP Purchase Plan and Phosphodiesterase explorer or bulk kits. For life science and drug discovery research use only. Not for diagnostic applications. IMAP Peptide Substrate Reconstitution Protocol for 50 nmoles vial (8000 test points at 100 nm) Reconstitute the 50 nmoles lyophilized substrate in 2.5 ml of 1x IMAP Reaction Buffer (as defined in the Screening Express and IPP package insert) to make a 20 µm fluorescent substrate solution. Vortex gently and invert vial to make sure that all of the lyophilized substrate goes into solution. You are now ready to prepare your substrate working solution for the IMAP assay. For example, if you are using a final reaction concentration of 100 nm substrate and want to add 5 µl of substrate solution per 20 µl reaction, then make a 4x working stock of 400 nm. (For 8000 x 20 µl reactions, this would be 800 µl of the 20 µm substrate solution plus 39.2 ml of Complete Reaction Buffer to make 40 ml of 400 nm substrate. The complete reaction buffer may contain 1mM DTT and other additions as directed by enzyme needs) IMAP Peptide Substrate Reconstitution Protocol for nmoles (50,000 test points at 100 nm) Reconstitute the nmoles lyophilized substrate in ml of 1x IMAP Reaction Buffer (as defined in the Screening Express and IPP package insert) to make a 100 µm fluorescent substrate solution. Vortex gently and invert vial to make sure that all of the lyophilized substrate goes into solution. Make a further 1/5 dilution of the substrate solution if you prefer a 20µM stock solution. (i.e., add 12.5 ml 1x IMAP Reaction Buffer to the 3.1 ml of 100 µm substrate to make 15.6 ml of the 20 µm substrate solution. Vortex gently to mix.) You are now ready to prepare your substrate working solution for the IMAP assay. For example, if you are using a final reaction concentration of 100 nm substrate and want to add 5 µl of substrate solution per 20 µl reaction, then make a 4x working stock of 400 nm. For 50,000 x 20 µl reactions, this would be 5 ml of the 20 µm substrate solution plus 245 ml of Complete Reaction Buffer to make 250 ml of 400 nm substrate. The Complete Reaction Buffer may be 1xIMAP Reaction Buffer containing 1mM DTT and other additions as directed by enzyme requirements. NOTES: Although most IMAP substrates are stable for up to two weeks at 4 C after reconstitution, we recommend that you aliquot any unused portion and store at -20 C. Avoid repeated freeze/thaw cycles Reconstituting the 50 nmoles substrate in 2.5 ml buffer will result in 1% BSA in this stock from the lyophilization and stablilization procedure. Running the assay at 100 nm substrate results thus in 0.005% BSA, which is very low and has been shown not to influence the assay results. However if you choose to run the assay at higher substrate concentrations, the concentration of BSA will increase proportionaly. BSA has the capability to bind to hydrophobic compounds and thus reduce their active concentration in the reaction which can lead to a right shift of IC50's. Please take this also in account when choosing your reaction buffer. Most IMAP buffer kits come with both, a BSA containing and a Tween containing reaction buffer. You might want to cosider as alternative to use the nmole/vial size, which is reconstituted to 100 µm with 1% residual BSA which results in a 5x lower residual BSA concentration as compared to the 50 nmoles size. IMAP Peptide Substrate 9

10 Table 2: Phosphodiesterase Substrates 20 nmoles substrate 120 nmoles substrate 50,000 Test Detection System (TR- FRET Tb donor: 1/400 for all) IMAP Progressive Binding System Original Binding System 8,000 Test Points 1 Points 1 based on Binding Minimum based on 100 nm 100 nm in 1x Buffer 1x Buffer Reagent Binding Binding Reagent Product Name Peptide Sequence 2,3 in reaction reaction A (%) B (%) Dilution Incubation Time Dilution camp PDE substrate 4 (FAM)-cyclic-(3,5 )-AMP R7505 R7506 FP /600 1 hour 1/400 TR-FRET /800 3h Not tested cgmp PDE substrate 4 (FAM)-cyclic-(3,5 )-GMP R7507 R7508 FP /600 1 hour 1/400 TR-FRET /800 4h Not tested 1: Number of test points is based on 100 nm substrate in a typical 384-well microplate assay consisting of a 20 µl PDE reaction 100 nm PDE substrate) and 60 µl of Binding Solution. 2: FAM = fluorescein; phosphodiesterase substrates are supplied as a 100 µm solution in 0.1% acetic acid. 3: TAMRA (tetramethylrhodamine) labeled substrates are also available. Please inquire. 4: Recommended storage for (FAM)-cyclic-(3,5 )-AMP and (FAM)-cyclic-(3,5 )-GMP, is at 20 C in the dark. Aliquots are recommended. Sales Offices Molecular Devices Corporation 1311 Orleans Dr. Sunnyvale, CA USA info@moldev.com USA UK Germany Japan Check our website for a current listing of our worldwide distributors. SOFTmax is a registered trademark and IMAP, Analyst and Acquest are trademarks of Molecular Devices Corporation. All other trademarks are property of their respective owners Molecular Devices Corporation. Printed in the U.S.A. R3475.S IMAP Peptide Substrate 10