
Qalabka N-Glycan
Sharaxaada Alaabta
Dukumeentigani waxa uu bixiyaa macluumaadka ku saabsan daryeelka guud iyo isticmaalka N-Glycan Kit ee siidaynta degdega ah enzymatic iyo calaamadaynta degdega ah ee N-glycans.Hab-maamuuska waxaa lagu ansaxiyay iyadoo la adeegsanayo unugyada difaaca jirka ee monoclonal waxaana sidoo kale lagu tijaabiyay in lagu sameeyo glycoprotein kale oo badan oo N-ku xiran.Waxaan kula talinaynaa isticmaalaha inuu xaqiijiyo sii daynta enzymatic ee muunaddooda gaarka ah.
Qaybaha
N-Glycan Kit (24T)
Module | Cawood leh | HCP0031A |
Deglycosylation Module HCP0031A-1 | IgG | 1.2 mg |
PNGase F | 45µL | |
PNGase F kaydinta | 0.5ml | |
Surfactant | 1.2mg*6 | |
Calaamadaynta Module HCP0031A-2 | budada reagent MS | 8.2mg*3 |
Anhydrous DMF | 1ml | |
Module-ka nadiifinta HCP0031A-3 | Elution Plate (96 ceel) | 1 gabal |
Elution Buffer | 5ml* 1 | |
Tusaalaha Diluent | 5ml* 1 | |
Tusaalaha Ururinta Module HCP0031A-4 | 8-tubo Strips (200μL) | 100 xabo |
8-koob Strips | 100 xabo | |
Saxan ururin (96 ceel) | 1 gabal | |
Saxan qashin ah | 1 gabal |
Baratakoolka
Isticmaaluhu wuxuu u baahan yahay inuu hoos u diyaariyo reagents iyo aaladaha ka hor intaadan bilaabin (Hal muunad IgG tusaale ahaan):
(1) 0.85% Nacl, 100µL;acetonitrile (darajada LC-MS) biyaha ultrapure;
(2) 15/85 = biyaha ultrapure / acetonitrile xal (v / v), 2mL;1/9/90=fomic acid/biyo ultrapure/acetonitrile(v/v) ,20ml;50mM ammonium formate,PH=4.4,500ml
(3) SPE vacuum bamka;96-si fiican u dheji processor-ka cadaadiska taban ama processor-ka cadaadiska togan;Qalabka kuleylka ama birta qubeyska (90 ℃ iyo 50 ℃);Isku-darka vortex; pipette.
Tallaabada 1: Deglycosylation degdeg ah
(1) Ku qas muunadda IgG oo leh 0.85% Nacl si aad u hesho diiradda ugu dambeysa ee IgG ee 2mg/mL.
(2) PNGase F kaydinta: ku milo 1 vial surfactant (1.2mg/3.6mg) leh (24μL/72μL)) PNGase F kaydinta, qas oo ku kaydi heerkulka qolka (xalkani wuxuu u baahan yahay in la diyaariyo kahor isticmaalka) .
(3) Ku dar 20μL IgG xalka 200μL tuubada falcelinta, ku dar 3μL PNGase F bakhaar, pipette kor iyo hoos si aad isugu qasto ka dibna ku dar 3.3μL biyo ultrapure ah, aspirate iyo u bixi si aad isugu qasto.
(4) Ku dar dhammaan xalka kor ku xusan moduleka kululaynta 90 ℃,3 daqiiqo, denature, ka dibna ka soo bax moduleka kuleylka, oo qabooji 3 daqiiqo heerkulka qolka.
(5) Ku dar 1.2μL PNGase F, aspirate oo bixi si aad isugu walaaqdo, kulayl 50 ℃, 5 daqiiqo oo leh moduleka kuleylka.Isticmaaluhu waxa uu diyaarin karaa calaamadaynta reagent inta uu sugayo.
(6) Ka soo bax moduleka kuleyliyaha, ka dibna ku qabooji 3daqiiqo heerkulka qolka.
Tallaabada 2. Calaamadaynta degdega ah ee Glycosylamines
(1) Xalka calaamadaynta reagent: ku milo hal dhalaal oo sumadeeya reagent (8.2mg/24.6m) DMF anhydrous(60.13μL/180.4μL) reagent ayaa si buuxda u milmay.
(2) Ku dar 6μL sumadeeyaha reagent ee tuubada falcelinta, damac oo bixi 5-10 jeer si aad u hubiso isku darka, ka dibna u oggolow falcelinta calaamadaynta inay ku sii socoto qolka.
heerkulka 5 daqiiqo.
(3) Ku dar 179 μL acetonitrile, ku dar tallaabada xigta.
Tallaabada 3: Nadiifinta Glycosyla oo lagu calaamadeeyaymiinooyinka
(1) Samee processor-ka cadaadiska taban ama processor-ka cadaadiska togan iyo saxan 96-well ah.
(2) Ceelasha xaaladda: ku dar 200μL biyo ultrapure ah saxanka sare u qaadista si aad u hagaajisid ceelasha, qashinka ku ururi saxaarada qashinka.
(3) Ceelasha siman: ku dar 200μL 15/85 biyo/acetonitrile xal (v/v) si loo siman yahay ceelasha, ku ururi qashinka saxaarad qashinka ah.
(4) Muunad raran: Ku shub muunada la qasi jiray acetonitrile, qashinkana ku ururi saxaarad qashin ah.
(5) Ceelasha ku maydh 600μL 1/9/90 formic acid/biyo ultrapure/acetonitrile(v/v) laba jeer ka dibna ku ururi qashinka saxaarad qashinka ah.
(6) Ka saar saxanka qashinka, oo u beddel saxanka qashinka.
(7)Ku maydh unugga 50μL elution baffer, glite glycans saxan ururinta, oo ku celi tallaabadan.
(8) Ku dar 200μL muunad dareere ah, isku dar.
Tallaabada 4. HILIC-FLR
(1) Tiirka Chromatography: ACQUITY UPLC® Glycan BEH Amide, 130 Å, 1.7 μm, 2. 1 x 150 mm (qaybta biyaha #186004742).
(2) Heerkulka tiirka: 60 °C
(3) Wajiga dareeraha A: 50 mM xal ammonium formate (darajada LC-MS ayaa lagula talinayaa), pH=4.4.
(4) Wajiga dareeraha B: 100% acetonitrile (darajada LC-MS waalagu taliyay).
(5) Heerka socodka: 0.4 ml/min;
(6) Hoos-u-dhac:
Waqtiga (min) | Heerka socodka (ml/min) | %A | %B | Qallooca |
0 | 0.4 | 25 | 75 | 6 |
35 | 0.4 | 46 | 54 | 6 |
36.5 | 0.2 | 100 | 0 | 6 |
39.5 | 0.2 | 100 | 0 | 6 |
43.1 | 0.2 | 25 | 75 | 6 |
47.6 | 0.4 | 25 | 75 | 6 |
55 | 0.4 | 25 | 75 | 6 |
(7) FLR hirarka dhererka: EX 265/EM 425 nm
(8) Heerka muunada FLR: 2 Hz
(9) Vol.: 10μL
Fadlan ogow in cabbirada sare ee ku salaysan mishiinka ku qalabaysan "ACQUITY® RDa"LCMS, isticmaaluhu wuxuu u hagaajin karaa cabbirka si waafaqsan qalabka kale.
Sdharbaaxo
Reagent | Kaydinta |
IgG | 2-8℃, 12 bilood |
PNGase F | -25 ~ – 15℃℃, 12 bilood, iska ilaali wareegyada qaboojinta/dhalaalaan |
PNGase F kaydinta | 2-8℃, 12 bilood |
Surfactant | Heerkulka qolka, 12 bilood |
MS reagent budada | 2-8 ℃, 12 bilood, ku hay mugdi |
Anhydrous DMF | Heerkulka qolka, 12 bilood |
Elution Buffer | Heerkulka qolka, 12 bilood |
Tusaalaha lagu qasi karo | Heerkulka qolka, 12 bilood |
96 saxan ellition ceel | Heerkulka qolka, marka la furo, ceelka aan la isticmaalin wuxuu u baahan yahay in lagu xidho balaastigga balaastigga ah oo lagu qalajiyo heerkulka qolka. |
200μL oo xariijimo 8 ah, xariijimo ah 8 koofi, 96 saxan ururinta ceelka, saxaarad qashinka | Heerkulka qolka, ururinta ceelasha 96 saxanka waa la xidhi karaa oo ku hay qallajiye heerkulka qolka |
Xusuusin
Badbaadadaada iyo caafimaadkaaga, fadlan xidho jaakad shaybaadhka iyo galoofyada.
Isticmaal talooyin nadiif ah mar kasta
Fadlan raac buug-gacmeedka si aad u kaydiso oo aad u isticmaasho reageyaasha