GE Q HP manuel d'utilisation

Instructions 71-7149-00 AN HiTrap ion exchange columns GE Healthcare HiTrap SP HP , 1 ml and 5 ml HiTrap Q HP , 1 ml and 5 ml HiT rap ™ SP HP an d Hi T rap Q HP a re pre pac ked , rea dy to us e cat io n and a nio n exc han ge co lum ns fo r met hod s cou tin g, gr oup s ep arat io ns, s amp le co nce ntra ti on an d sam ple c lea n-u p of ch arg[...]

p. 2 Code No. Designation No. su pplied 17 - 1151 - 01 Hi T rap S P HP 5 x 1 ml 17 - 1152-01 HiTrap SP HP 5 x 5 m l 17 - 1153- 01 Hi T rap Q H P 5 x 1 ml 17 - 1154 -01 Hi T rap Q H P 5 x 5 ml Connectorkit Connectors supplied Usage No. su pplied 1/16” mal e/ lu er fem al e Co nne ct io n of syr in ge to to p of HiTr ap column 1 T ub ing c onn ec t[...]

p. 3 1. Description Media properties SP Seph arose ™ H igh P er for ma nce an d Q Sep ha rose H igh P er fo rma nce are s tr ong ca ti on an d st ron g ani on ex cha nge rs re sp ec tiv ely . Bot h rem ain cha rge d and m ain tai n high c apa cit y over b roa d pH ra nge s. T he f unc ti ona l gro ups a re cou ple d to th e ma tr ix vi a chem ica[...]

p. 4 Ta b l e 1 . Hi T r ap SP HP a nd Hi T rap Q HP c olu mns c hara ct eri st ic s Co lum n vol ume s 1 ml o r 5 ml Co lum n dim ens io ns 0.7 × 2.5 c m (1 ml ) and 1 .6 × 2.5 c m (5 m l) T ota l ion ic ca pac ity 0.14– 0.20 m mo l (C l – )/m l me di um (Q) 0.15– 0. 20 mm ol ( H + )/ml mediu m (SP ) Dy nam ic bi ndi ng ca pa city SP: a pp[...]

p. 5 2. Selection of ion exchanger and conditions Io n exch ang e chr oma tog rap hy is b ase d on a dso rpt io n and re ver sib le bi ndi ng of ch arg ed sa mpl e mo lec ule s to op pos ite ly ch arg ed gro ups at ta che d to an in so lub le ma tr ix . The p H val ue at w hic h a bio mol ecu le ca rr ies no n et cha rge i s cal led t he i soe lec [...]

p. 6 Strategy 1. Binding and elution of all sample components Bi ndi ng is a chi eved b y cho os ing a s tar t b uf fe r wit h a lo w pH for H iTrap SP HP or a h igh p H for Hi Trap Q HP . T he io nic s tre ngh t sho uld b e kep t as lo w as po ssi bl e to all ow al l com po nent s to b in d to th e ion ic ex chan ge (< 5 mS/cm ) . Th is res ult[...]

p. 7 Th e buf fe ri ng io n sho uld c arr y th e sam e cha rge a s the i on ex cha nge g rou p an d sho uld h ave a pK a wit hin 0 .5 pH u nit s of t he pH u se d in th e sep ara ti on. Bu f feri ng io ns of o pp osi te cha rge m ay ta ke pa r t in th e ion e xch ang e pro ces s and caus e local distu rbance s in pH. Starting pH Cation excha ngers [...]

p. 8 Ta b l e 3 . Buf fers for anion exchange chromatography. pH in ter va l Sub st anc e Co nc . (mM ) Co unte r-io n pK a (25 ° C ) 1 4.3–5.3 N-Methylpiperazine 20 Cl - 4. 75 4.8–5.8 Piperazine 20 Cl - or HC OO - 5.33 5. 5– 6.5 L-His ti din e 20 Cl - 6.04 6.0– 7 .0 bis- T ris 20 Cl - 6.48 6. 2– 7.2; 8. 6– 9.6 bi s-T r is p rop an e 2[...]

p. 9 Fig 3. Recommended buffer substances for anion exchange chromatography. Th e colu mn s can be o pe rate d wi th a sy rin ge, p eri st alt ic pu mp o r a chromatography system. 3. Operation Buff er preparation Wate r and c hem ica ls us ed fo r buf fe r pre par at ion s hou ld be o f hig h pur ity . It is re com men de d to f ilte r th e buf f[...]

p. 10 Ta b l e 4 . Prep ack ed col umn s for d esa lt ing a nd bu f fer e xcha ng e. Code No Column Loading volume Elution volume Comments Application 17 - 1408 -01 Hi T rap 0.1– 1. 5 ml 1. 3– 4.0 m l Pr epa cke d wit h For de sa lti ng an d bu f fer De sal ti ng Se pha de x™ G-25 exch ang e of p rote in Super f ine. Requires ex tract s (M r [...]

p. 11 Purifi cation 1. Fi ll th e syr ing e or p ump tu bin g wi th s tar t b uf fer ( lo w io nic s tre ngt h ). Rem ove t he s top per a nd co nne ct t he co lum n to th e syr ing e ( wit h th e pro vid ed a dapt or) , “dro p to dro p” to av oid i ntr odu ct ing a ir in to th e colum n. 2. Rem ove t he sn ap -of f e nd at t he co lum n out l[...]

p. 12 Note: If a P1-pu mp is u sed a m ax f lo w rat e of 1 –3 ml/m in ca n be r un on a Hi T r ap 1 ml co lu mn pa cked w it h Sep har ose H igh P er for ma nce media. 4. Optimization If sa mp le com po sit ion i s unk no wn, a s imp le sc reen ing t es t wit h th e aid of a sy rin ge or p ump c an be p er fo rm ed to o ptim ize s ta r tin g pH [...]

p. 13 Further optimization Th e reco mme nd ati ons g ive n abo ve wi ll gi ve a sou nd b asi s for de vel opi ng a n ef f ici ent p uri f ica tio n st ep. De tai ls of h ow f low r ate, s amp le lo ad ing , par t icl e si ze and e lut io n sch eme m ay be o pti miz ed to m eet t he s pec ial n eed s can be fo und i n th e han dbo ok , Io n Exc han[...]

p. 14 Elution with stepwise ionic strength gradients Step wi se el uti on i s the s equ ent ia l use o f the s ame b uf fe r at di f fere nt io nic st ren gt hs. I t is te chn ical ly si mpl e and f as t, an d is su ita ble f or syr in ge op era tio n. I t is of ten u se d for s amp le co ncen tra tio n and s am ple cl ea n-u p. Step wis e el uti o[...]

p. 15 – A p eri st alt ic pu mp an d a gra die nt mix er e.g . pu mp P- 1, gra die nt mix er GM- 1 . – A o ne pu mp sy ste m, e.g . ÄK T A pr ime ™ plus. – A t wo pu mp sy st em, e. g FPLC or ÄK TA. 1. Choose s tarting conditions as outlined under Optimizing star ting conditions. 2. Equilibr ate the column, see Purif ication. 3. Ad jus t [...]

p. 16 Fig 4. Bind in g cap aci ty of hu man I gG , HSA an d hum an t ran sfe rr in at d if fe rent p H´s on Hi Trap Q HP , 1 m l. 4. Ap pl y the s amp le s olu tio n to th e col umn w it h a pum p or a syr in ge, at a f low r ate e qua l to th e fl ow ra te to b e use d in t he pu rif i cat ion m et hod . Col le ct f rac ti ons a nd co nti nue s a[...]

p. 1 7 7. Scaling up For q uic k scal e- up of pu ri f icat io ns ( bac k pre ssu re wil l inc rea se ), t wo o r th ree Hi T rap i on ex chan ge co lum ns of t he sa me ty pe ca n be co nne ct ed in s er ies . For f ur t her s cale -u p SP Se pha ros e High P er fo rma nce a nd Q Se pha ros e Hig h Per f orm anc e are av ail abl e in pr epa cke d [...]

p. 18 Rel ate d pro duc t s No. S upp lie d Co de No . Hi T rap I EX Se le ct ion K it 7 x 1 ml 17 - 60 02- 33 Hi T rap D es alt in g 5 x 5 ml 17 - 1408 -01 Hi T rap D es alt in g 100 x 5 m l* 11-0 003 -29 HiP rep 26 /10 Desa lt ing 1 x 53 ml 17 -5087-01 HiP rep 26 /10 Desa lt ing 4 x 53 ml 17 -5087-02 PD- 10 Desalti ng column 30 1 7 - 0851 - 01 Hi[...]

p. 19 Realated literature Ion Exchange Chr omatography & Chromatofocusing Handbook, Principles and Methods 1 11 - 0004-21 Io n Exc han ge Co lu mns a nd Me di a, Sel ec ti on Gu ide 1 18 - 1127 -3 1 Co nven ien t Prot ein P uri f ica tio n, Hi T rap C olu mn Gui de 1 18 - 1129 -81[...]

HiTrap, Sepharose, Sephadex, FPLC, ÄKT A , ÄKT Aprime, ÄKTAdesign, Dr op Design, NICK, NAP , HiPrep and HiLoad are trademarks of GE Healthcare companies. GE, imagination at work and GE monogram are trademarks of General Electric Company. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthca[...]