Simplified TA nanoDSC III protocol STOP This protocol is for qualified users operating nanoDSC III in IBC 402 only. Dr. Jao accepts no responsibility for actions taken as a result of using this protocol. Reading the manufacturer's User’s Manual is highly recommended. Sample preparation: 1. For e a c h c ell, pr e p are >650 µL (700 µL is e a si er for lo a din g) of solution, d e g a ss e d a t st artin g t e m p er a t ur e for at l ea st 10 min. un d er a va c u u m of 500 m m-Hg. 2. Prot ein c o n c e ntr ations ar e usu ally 0.2 m g / mL t o 1 m g / mL. 3. Pre p ar e 30-50 mL of b uff er for b as elin e s c a ns a n d cl e a nin g p u rp os e. 4. Ple as e c o nsult m a n a g er if yo u int e n d t o us e n o n- a q u e o us solutions. Start up: 5. Pow er u p th e instru m e n t if th e instru m e n t is n ot o n. 6. Boot u p th e c o ntrol PC a n d lo g o n t o Wind o ws. 7. M ak e sur e th e int erne t is n ot c o n n e c t e d th e n d o u bl e click “M e as ur e m e nt a n d Aut o m a tio n” ic o n t o est a blish c o n n e c tion b e t w e e n c o ntrol PC a n d th e instrum e nt. 8. Initi alize DSC c o ntrol softw ar e by d o u bl e-clickin g “La u n c h DSCRun”. Th e softw ar e c o n t ains thre e t a bs: Experiment Method, Monitor, a n d Data. 9. Sho ul d th e pr o gr a m fail to st art, clos e th e win d o w a n d try a g ain. 10. Basic p ar a m e t ers for a DSC ex p erim e nt m a y b e s et u p by lo a d in g a pr eviously s av e d ex p erim e nt m e t h o d (. ds c cf g). Sample loading and start: 11. Dr ain w a t er fro m r efer e n c e c ell, rins e th e c ell twi c e with yo ur b uff er a n d dr ain residu al b uff er. 12. Lo a d b uff er ( d e g asse d ) t o th e c ell c ar efully by a o n e- mL pipe t t e: a . Att a c h o n e e n d of th e c a pillary c ell with a pi p e tt e tip. b . With dr a w 650 µL of d e g a ss e d b uff er a n d a tt a c h th e pi p e tte t o th e ot h er e n d. c . Slowly disp e ns e b uffer t o th e c a pillary c ell until th e s olution e m er g es fro m th e o utl et. d . G e ntly os cillat e 3 times t o r e m ov e b u b bl e s. e . Slowly with dr a w ex c ess b uff er so th a t o nly 5m m in h ei ghts of solution c a n b e s e e n a t th e ot h er o utl et. Protocol adopted from NanoDSC Getting Started Guide M101SP.pdf Page 1/3 02/01/2012 f. D et a c h th e pi p e tt e (with pi p e tt e tip) witho ut disturbin g th e s olution. g. D et a c h th e ot h er pi p e t t e tip slowly. h. Fill bl a ck vinyl c a p with b uff er a n d c a p o n e e n d of th e c a pill ary c ell. i. Fill s a m pl e c a pill ary with s a m e pro c e d ures d es crib e d a b o v e a . t o h. 13. Tight e n th e pr essur e h a n dl e. 14. Within th e DSCRun c o ntrol softw ar e, m o n itor th e h e a t c h a n g e o n th e Monitor p a g e . Alt er th e plottin g s c al e t o b e -15 to 20 µW a n d w ait for th e b as elin e t o st a bilize within 1 µW. 15. While o n th e Monitor p a g e , pr essurize th e c ell to 3 at m by clickin g t h e pr essurize b utt o n (Up arro w). W at c h clos ely on th e c h a n g e of th e h e a t b as elin e. Su d d e n c h a n g e of th e b as elin e >10 µW in di c a t e s bu b bl es in th e c ells a n d th e c ells n e e d t o b e r efille d. 16. Set u p ex p erim e nt p ar a m e t ers a n d st art a s et of 10 s c a ns for c o n ditionin g t h e c ells ov erni ght. Ple a s e n ot e th a t a t e m p er a t ur e s c a n of 5-110 ℃ is usu ally suffici e nt for m ost prot ein s a m pl es. N ev e r fre ez e yo ur s a m pl e in fille d c ells. An e q uilibr ation p erio d of 900 s e c o n ds is re c o m m e n d e d for e a c h s c a n. 17. Aft er a n ov ernight exp erim e nt of b uff er/ b uff er s c a ns, c h e ck for b as elin e re p e a t a bility. If th e b a s elin e is st a bl e, st o p th e c urr e nt c o n ditionin g ex p erim e nt a n d w ait for th e t e m p er a t ur e t o c o m e t o idl e t e m p er a t ur e b efor e d e pr essurizin g th e c e lls. 18. Refill b ot h c ells with d e g a ss e d b uff er a n d st art a bl a nk ex p erim e nt for on e s c a n usin g th e s a m e p ar a m e t ers as for the c o n ditionin g ex p erim e nt b ut a differ e nt filen a m e. 19. Wh e n th e c ell t e m p e rat ur e c o m es d o w n to th e idl e t e m p er a t u re, r efill ref er e n c e c ell with b uff er a n d r e pl a c e s a m pl e c ell with yo ur sa m pl e s olution ( d e g ass e d) b y followin g instru ctions of lo a din g pro c e d ur es 12 a . ~12 h, ex c e p t for th e bl a ck c a p for s a m pl e c a pillary whic h sh o ul d b e fille d with s a m pl e solution b ef or e c a pin g. 20. Tight e n th e pr essur e h a n dl e. 21. St art o n e s c a n usin g th e s a m e p ar a m e t ers as for th e bl a nk s c a n a n d pr ovid e th e d a t a a differ e nt filen a m e . 22. O n c e t h e t e m p er a t u re c o m es d o w n t o idle , d e pr essurize c ells a n d pro c e e d t o th e cl e a nin g pro c e d ur e. Protocol adopted from NanoDSC Getting Started Guide M101SP.pdf Page 2/3 02/01/2012 Cleaning Procedure: The followin g is a d o p t e d fro m N a n o DSC G e ttin g St art e d G ui d e . 23. With dr a w yo ur s a m ple fro m s a m pl e c ell using 1-mL pip e tt e a n d c h e ck for pr e ci pit at es. Rins e s a m pl e c ell with 1mL buff er thr e e tim es with pi p e tt e. 24. Em pty b ot h c ells a n d r efill c ells with 15% D e c o n 90 d e t er g e n t solutio n. C a p c a pill ary c ells, inst all pr essur e h a n dl e a n d p erform o n e s c a n c ycl e fro m 50 t o 80℃ at 1℃/ min. 25. Wh e n th e t e m p er a tur e c o m es d o w n t o idl e, d e pr essurize an d re m ov e pr essur e h a n dl e a n d bl a ck c a ps. 26. Clos e th e DSCRun c o ntrol pro gr a m. Em p ty d e t er g e nt solution fro m b ot h c ells a n d p ass 1L d dH2O throu g h c ells usin g flushin g e q ui p m e nt a n d TA’s d e g assin g st ation as sh o w n b elo w. 27. Wh e n d o n e, a c tiv at e DSCRun c o ntrol softw ar e. 28. Re p e a t st e p 24 t o 26 ex c e p t r e pl a cin g d e t er g e n t solution with 50% Formi c a ci d. 29. Aft er p assin g 2L d d H2O thro u g h b ot h c ells, th orou g hly cl e a n all ar e a t h a t mi g ht h a v e b e e n c o nt a min a t e d by cl e a nin g solutions. 29. A ctiv at e DSCRun c o ntrol softw ar e a n d fill b ot h c ells with d e g a ss e d d dH2 O. 30. P erform 10 w a t er / w a t er s c a ns fro m 10 t o 110 ℃ at 1℃ / min t o c h e ck for b as elin e st a bility. 31. Wh e n th e c ells ar e cle a n, d e pr essurize c ells a n d le a v e th e instru m e nt o n. 32. Turn off c o ntrol c o m p u t er a n d m o nit or. 33. For a n alysis, pl e as e ref er t o th e NanoAnalyze Software Guide. R S Protocol adopted from NanoDSC Getting Started Guide M101SP.pdf Page 3/3 02/01/2012