This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.

Transcription

1 Idd U Th TrkA Ka Aay/Ihbor Scrg K dgd o maur h ac of rcomba caalyc doma of TrkA for h rapd ad aluao of hbor or acaor. Th phophoyro pcfc moocloal abody ud h aay k ha b dmorad o rcogz h phophoyro rdu rcomba Tyro ka-ubra-1, whch ffcly phophorylad by TrkA ro. Applcao for h k clud: 1) Scrg hbor or acaor of rcomba caalyc doma of TrkA. 2) Dcg h ffc of pharmacologcal ag o rcomba caalyc doma of TrkA. Th aay k for rarch u oly ad o for u dagoc or hrapuc procdur. Sorag Upo rcp or h ATP a -20 C. Upo rcp or all ohr compo a 4 C; Do o xpo rag o xc lgh

2 Iroduco Th Trk (ropomyo rcpor ka) rcpor blog o h famly of rcpor yro ka, ad hr rk g ha b dfd mammal. Th TrkA prooocog wa fr dfd a a r growh facor rcpor (NGFR) (1, 2), followd by TrkB ad TrkC (3). Nr growh facor (NGF) h prfrrd lgad for TrkA, bra-drd urorophc facor (BDNF) ad Nuroroph-4/5 (NT-4/5) ar prfrrd for TrkB, ad Nuroroph-3 (NT-3) for TrkC (3). Th pcfc ar o abolu, ad NT-3 alo a lgad for TrkA ad TrkB. Th Trk rcpor ar rammbra glycopro of ~140 kd. Thy ar yro ka wh a xracllular lgad-bdg doma coag mulpl rpa of luc-rch mof, wo cy clur (C1, C2), wo mmuoglobul-lk doma (Ig1, Ig2), ad a gl rammbra doma (4). Th yro ka doma ar hghly rlad (~80% amo acd dy), howr h xracllular doma ar mor drg (~30%). Sgalg hrough h Trk rcpor-h ma pahway. Nuroroph bdg o Trk rcpor rggr dmrzao ladg o h acao of dffr galg pahway hrough rcrum of arou adapr molcul. Th ma bdg for Trk ubra ar wo yro rdu ha, o acao of h Trk rcpor, bcom phophorylad. Two complx of adapr molcul bd o h yro rdu locad h juxammbra rgo of h Trk rcpor, h Shc/Grb2/SOS ad h FRS2/SHP-2/Grb2/SOS complx. Ra/Raf/MEK/MAPK duc h dffrao of uro ad ur growh (5). Maurm of TrkA Ka acy Th proocol grally rgardd a mo for h quaa maurm of TrkA ka acy ol cubao of h TrkA ka ampl wh ubra, hr a aural or yhc polyppd (uch a poly[glu,tyr]4:1), h prc of Mg 2+, M 2+ ad 32 P-labld ATP. Th raco rmad by "pog" a ampl oo a flr papr dc, followd by mmro acd o prcpa h radolabld produc. Th flr papr ar h wahd xly o rmo ucorporad radolabl ad h radoacy coud. Whl, h mhod labor-, gra hazardou radoac wa, ad dpd o a radooop of hor half-lf. I parcularly uuabl wh ka aay ar oly prformd o a frqu ba. Th TrkA Ka Aay/Ihbor Scrg K u a horradh proxda coupld a-phophoyro moocloal abody a a rporr molcul a 96-wll ELISA forma. Th aay prod a o-oopc, ad pcfc mhod o dc ka acy of rcomba TrkA caalyc doma.

3 Prcpl of h Aay Th TrkA Ka Aay/Ihbor Scrg K a gl-, o-quaa mmuoaay for ka acy of rcomba caalyc doma of TrkA. Pla ar pr-coad wh a wly dgd Tyro ka-ubra-1, whch ca b aly phophorylad by rcomba caalyc doma of TrkA. Th dcor abody PY-39, a abody ha pcfcally dc h phophoyro rdu o Tyro ka-ubra-1. Th TrkA Ka Aay/Ihbor Scrg K mgh b ud o follow h kc of rcomba caalyc doma of TrkA a wll a crg TrkA hbor or acaor. To prform h, h rcomba caalyc doma of TrkA dlud Ka Buffr, ppd o h wll ad allowd o phophoryla Tyro ka-ubra-1 o h wll h prc of Mg 2+, M 2+ ad ATP. Th amou of phophorylad Tyro ka-ubra-1 maurd by bdg wh a horradh proxda cojuga of PY-39, a a-phophoyro moocloal abody, whch h caalyz h coro of h chromogc ubra ra-mhylbzd (TMB) from a colorl oluo o a blu oluo (or yllow afr h addo of oppg rag). Th color quafd by pcrophoomry ad rflc h rla amou of TrkA ka acy h ampl. For kc aaly, h rcomba caalyc doma of TrkA addd o h wll a mlar faho ad a aryg m h raco oppd by h addo of a chlaor, odum hyldamraaca (EDTA) ad h amou of phophorylad ubra drmd a bfor. Th TrkA Ka Aay/Ihbor Scrg K dgd o drm o-oopc kc aaly of h TrkA caalyc doma ka acy. Carful ao o oprao ad h aay proocol wll prod h gaor wh a rlabl ool for h aluao of hbor or acaor of TrkA ka.

4 Summary of Procdur Add 100 µl of raco mxur o h wll Wah h wll Icuba for 60 m a 30 C Add 100 µl of HRP cojugad a-phophoyro abody Wah h wll Icuba for 60 m a room mp. Add 100 µl of Subra Rag Add 100 µl of Sop Soluo Maur aborbac a 450 m

5 Maral Prodd All ampl ad adard hould b aayd duplca. Th followg compo ar uppld ad ar uffc for h o 96-wll mcror pla k. Mcropla: O mcropla uppld rady o u, wh 96 wll (12 rp of 8-wll) a fol, zp-lock bag wh a dcca pack. Wll ar coad wh rcomba Tyro ka-ubra X Wah Buffr: O 100 ml bol of 10X buffr coag 2%Tw -20. Ka Buffr: O 20 ml bol of 1X buffr ud for Ka Raco Buffr ad ampl dluo. 20X ATP: Lyophlzd ATP Na 2 al. Rcou co of al wh 1.6 ml of H 2 O. Mx gly ul dold. Fal cocrao of ATP hould b 1.25 mm ATP. Th ATP oluo ca b ord mall alquo (.g. 100 µl) a -20 C. Th 1 mm ATP ock oluo mu b dlud o 62.5 µm Ka Raco Buffr a h m of h aay. HRP cojugad Dco Abody: O bol coag 12 ml of HRP (horradh proxda) cojugad a-phophoyro moocloal abody (PY-39). Subra Rag: O bol coag 12 ml of h chromogc ubra, ra-mhylbzd (TMB). Rady o u. Sop Soluo: O bol uppld rady o u, coag 12 ml of 1.25 N H 2 SO 4. Rady o u.

6 Maral Rqurd bu o Prodd TrkA Po Corol: O al coa 100 u rcomba caalyc doma of TrkA. Th Po Corol hould b addd o h fr wll a 1 u/wll. For ac, h ca of 100 u/100 µl TrkA Po Corol, dlud Po Corol 1:10 wh Ka Buffr, u 10 µl for 1 aay. Th Po Corol paraly dlrd wh a dry c from ohr k compo. Th Po Corol hould b ord alquo a blow -70 C. Aod rpad frz ad haw. 10X Sauropor (100 M): Sauropor aalabl from Sgma, Ca#. S µm ock oluo (DMSO) dlud 1:100 Ka Buffr. 10X Qurc dhydra: 500 µm Qurc dhydra (Sgma Ca# Q-0125: mak 10 mm DMSO oluo ad dlud 1:20 war) Ppor: 2-20 µl, µl ad µl prco ppor wh dpoabl p. Prco rpag ppor. Wah bol or mulchal dpr for pla wahg. Mcrocrfug ad ub for ampl prparao. Vorx mxr. Pla radr capabl of maurg aborbac 96-wll pla a dual walgh of 450 m/540 m. Dual walgh of 450/550 or 450/595 m ca alo b ud. Th pla ca alo b rad a a gl walgh of 450 m, whch wll g a omwha hghr radg. Rag rror. Dozd war of h hgh qualy.

7 Prcauo ad Rcommdao Sor h ATP a -20 C alquo. Sor all ohr compo a 4 C. Do o xpo rag o xc lgh. Aod frz/haw cycl. Allow all h compo o com o room mpraur bfor u. Do o u k compo byod h dcad k xprao da. U oly h mcror wll prodd wh h k. R all drg rdu from glawar. U dozd war of h hgh qualy. Do o mx rag from dffr k. Th buffr ad rag ud h k coa Kaho-CG a prra. Car hould b ak o aod drc coac wh h rag. Do o mouh pp or g ay of h rag. Do o mok, a, or drk wh prformg h aay or ara whr ampl or rag ar hadld. Huma ampl may b coamad wh fcou ag. Do o g, xpo o op woud or brah arool. War proc glo ad dpo of bologcal ampl proprly. Dpo of ra-mhylbzd (TMB) coag oluo complac wh local rgulao. CAUTION: Sulfurc Acd a rog acd. War dpoabl glo ad y proco wh hadlg Sop Soluo.

8 Dald Proocol Th TrkA Ka Aay/Ihbor Scrg K prodd wh rmoabl rp of wll o h aay ca b carrd ou o para occao ug oly h umbr of rp rqurd for h parcular drmao. Du o chcal rao, h k adjud o maur ka acy of h rcomba caalyc doma of TrkA (TrkA Po Corol), whch hould b ud all aay. Dpoabl pp p ad rag rough hould b ud for all lqud rafr o aod cro-coamao of rag or ampl. Prparao of Workg Soluo 1. Prpar a workg oluo of Wah Buffr by addg 100 ml of h 10X Wah Buffr (prodd) o 900 ml of dozd war. Mx wll. 2. Prpar Ka Raco Buffr (ATP plu) by mxg followg rag. Ka (prodd) 20X ATP (prodd) aay aay aay Buffr 9.5 ml 950 µl 95 µl 0.5 ml 50 µl 5 µl Toal 10 ml 1000 µl 100 µl *You wll d µl of Ka Raco Buffr (ATP plu) pr aay wll. Mx wll. Dcard ay uud Ka Raco Buffr (ATP plu) afr u. Sadard Aay 1. Rmo h appropra umbr of mcror wll from h fol pouch ad plac hm o h wll holdr. Rur ay uud wll o h fol pouch, rfold, al wh ap ad or a 4 C. 2. Prpar h Ka Aay Buffr coag chmcal ad yro ka hbor. All aay hould b do duplca. 3. Add 10 µl of 0.1 u/µl TrkA Po Corol or ral dluo of TrkA Po Corol o h wll of h aay pla o c.

9 4. Bg h ka raco by addo of 90 µl Ka Raco buffr pr wll, cor wh pla alr, ad cuba a 30 C for 60 mu. 5. Wah wll f m wh Wah Buffr makg ur ach wll flld complly. Rmo rdual Wah Buffr by gl appg or aprao. 6. Pp 100 µl HRP cojugad Dco Abody PY-39 o ach wll, cor wh a pla alr ad cuba a room mpraur for 60 mu. Dcard ay uud cojuga. 7. Wah wll f m wh Wah Buffr makg ur ach wll flld complly. Rmo rdual Wah Buffr by gl appg or aprao. 8. Add 100 µl of Subra Rag o ach wll ad cuba a room mpraur for 5 15 mu. 9. Add 100 µl of Sop Soluo o ach wll h am ordr a h prouly addd Subra Rag. 10. Maur aborbac ach wll ug a pcrophoomrc pla radr a dual walgh of 450/540 m. Dual walgh of 450/550 or 450/595 m ca alo b ud. Rad h pla a 450 m f oly a gl walgh ca b ud. Wll mu b rad wh 30 mu of addg h Sop Soluo. No-1: Compl rmoal of lqud a ach p al o good prformac. Afr h la wah, rmo ay rmag Wah Buffr by aprag or dcag. Ir h pla ad blo aga cla papr owl. No-2: Rlabl gal ar obad wh hr O.D. alu do o xcd 0.25 u for h blak (o zym corol), or 2.5 u for h TrkA po corol. No-3: If h mcropla radr o capabl of radg aborbac grar ha h aborbac of h W1 po corol, prform a cod radg a 405 m. A w O.D. alu, maurd a 405 m, ud o drm TrkA acy of off-cal ampl. Th radg a 405 m hould o rplac h o-cal radg a 450 m.

10 Kc Aay 1. Rmo h appropra umbr of mcror wll from h fol pouch ad plac hm o h wll holdr. Rur ay uud wll o h fol pouch, rfold, al wh ap ad or a 4 C. 2. Prpar all ampl (dlud wh Ka Buffr a dd). All ampl hould b aayd duplca. 3. Add 10 µl of 0.1 u/µl TrkA Po Corol or ral dluo of TrkA Po Corol o h wll of h aay pla o c. 4. Bg ka raco by addo of 90 µl Ka Raco Buffr duplca pr wll md ral (uggd ral 5 mu bu hould b ddually drmd for ach ym). Afr h fal addo, cuba a 30 C for 20 mu. 5. Sop h raco by flckg ou h co. (Alraly, h raco may b rmad by h addo of 150 µl 0.1 M Na EDTA, ph 8.0 o ach wll). 6. Wah wll f m wh Wah Buffr makg ur ach wll flld complly. Rmo rdual Wah Buffr by gl appg or aprao. 7. Pp 100 µl HRP cojugad Dco Abody PY-39 o ach wll, cor wh a pla alr ad cuba a room mpraur for 60 mu. Dcard ay uud cojuga. 8. Wah wll f m wh Wah Buffr makg ur ach wll flld complly. Rmo rdual Wah Buffr by gl appg or aprao. 9. Add 100 µl of Subra Rag o ach wll ad cuba a room mpraur for mu. 10. Add 100 µl of Sop Soluo o ach wll h am ordr a h prouly addd Subra Rag. 11. Maur aborbac ach wll ug a pcrophoomrc pla radr a dual walgh of 450/540 m. Dual walgh of 450/550 or 450/595 m ca alo b ud. Rad h pla a 450 m f oly a gl walgh ca b ud. Wll mu

11 b rad wh 30 mu of addg h Sop Soluo. Rcommdao Spcal codrao wh crg acaor ad hbor I ordr o ma h hbory ffc o TrkA acy h chmcal corrcly, cary o coduc h corol xprm of Sol corol a la oc for ry xprm ad Ihbor corol a la oc for h fr xprm, addo o T ampl, a dcad h followg abl. Wh chmcal cau a hbory ffc o TrkA acy, h ll of A450 wakd a compard wh Sol corol. Th hgh ll of A450 o obrd Ihbor corol (uually A450<0.4). Aay rag Ka Raco Buffr 10X Ihbor or qual Sol for Ihbor 10X Sauropor (100 M)* TrkA Po Corol (0.1 u/µl)** T ampl Sol corol Ihbor corol 80 µl 80 µl 80 µl 10 µl µl µl 10 µl 10 µl 10 µl * 10X Sauropor (100 M): S co Maral Rqurd bu o Prodd ** TrkA Po Corol (0.1 u/µl)**: S co Maral Rqurd Prodd 1. Followg h abo abl, add h Rag o ach wll of h mcropla. Fally, a raco by addg 10 µl of Dlud TrkA Po Corol o ach wll ad mxg horoughly a room mpraur. Cor wh pla alr. Icuba a 30 C for 60 mu. 2. Follow h Sadard Aay, p 5-10.

12 Ealuao of Rul 1. Arag h aborbac alu for h TrkA ampl duplca (Po Corol) ad all xprmal ampl duplca alu (wh applcabl). Wh h TrkA po corol (1 u/aay) cludd a a ral corol for h phophorylao raco, h aborbac alu hould b grar ha 1.2 wh a backgroud l ha For kc aaly, o graph papr, plo h ma aborbac alu for ach of h m po o h Y-ax ru h m of ach raco (mu) o h X-ax. Aay Characrc Th TrkA Ka Aay/Ihbor Scrg K ha b how o dc h ka acy of rcomba caalyc doma of TrkA. Th aay how good lary of ampl rpo. Troublhoog 1. Th TrkA po corol hould b ru duplca, ug h proocol dcrbd h Dald Proocol. Icubao m or mpraur gfcaly dffr from ho pcfd may g rroou rul. 2. Th raco cur arly a ragh l f h kc of h aay of h fr ordr. Varao h proocol ca lad o o-lary of h cur, a ca aay kc ha ar ohr ha fr ordr. For a o-lar cur, po o po or quadrac cur f mhod hould b ud. 3. Poor duplca, accompad by lad alu for wll coag o ampl, dca uffc wahg. If all ruco h Dald Proocol wr followd accuraly, uch rul dca a d for wahr maac. 4. Orall low gal may dca ha dccao of h pla ha occurrd bw h fal wah ad addo of Subra Rag. Do o allow h pla o dry ou. Add Subra Rag mmdaly afr wah.

13 Rag Sably All of h rag cludd h TrkA Ka Aay/Ihbor Scrg K ha b d for ably. Rag hould o b ud byod h ad xprao da. Upo rcp, k rag hould b ord a 4 C, xcp h TrkA Po Corol mu b ord a blow -70 C. Coad aay pla hould b ord h orgal fol bag ald by h zp lock ad coag a dcca pack. For rarch u oly, o for u dagoc or hrapuc procdur.

14 Exampl of T Rul Fg.1 Do dpdcy of rcomba TrkA caalyc doma zym raco A ATP + ATP TrkA do (u ) Fg.2 Tm cour of rcomba TrkA caalyc doma zym raco A Tm (m.)

15 Fg.3-1 Do dpdcy of ATP (rcomba TrkA caalyc doma) A ATP coc. (um) Fg.3-2 Km for ATP (rcomba TrkA caalyc doma) y = x R 2 = Km 9.4(uM) > / < <>

16 Fg.4-1 Effc of broad-pcrum ka hbor auropor o acy of rcomba TrkA caalyc doma zym raco. Fg.4-2 Effc of broad-pcrum ka hbor auropor o acy of rcomba TrkA caalyc doma zym raco ug radooop gamma- 32 P-ATP Sau ropor c o c.(m) Sau ropor c o c.(m) Sau ropor c o c.(m) Sau ropor c o c.(m) R l a I y ( % ) R l a I y ( % ) R l a I y ( % ) R l a I y ( % ) Sauropor co c.( M) Sauropor co c.( M) Sauropor co c.( M) Sauropor co c.( M) R l a I y ( % ) R l a I y ( % ) R l a I y ( % ) R l a I y ( % )

17 Rfrc 1. Kapla, D.R., Hmpad, B.L., Mar-Zaca, D., Chao, M.V., ad Parada, L.F. Th rk proo-ocog produc: A gal raducg rcpor for r growh facor. Scc 252: , Kl, R., Jg, S.Q., Nadur, V., O'Rourk, E., ad Barbacd, M. Th rk proo-ocog cod a rcpor for r growh facor. Cll 65: , Barbacd, M. Th rk famly of uroroph rcpor. J. Nurobol. 25: , Schdr R, Schwgr M. A ol modular moac of cll adho mof h xracllular doma of h urogc rk ad rkb yro ka rcpor. Ocog. 6: Kapla, D.R. ad Mllr, F.D. Nuroroph gal raduco h rou ym. Curr. Op. Nurobol. 10: , 2000.