N-Glycan Kit
Product Description
Cov ntaub ntawv no muab cov ntaub ntawv hais txog kev saib xyuas dav dav thiab kev siv N-Glycan Cov Khoom Siv rau kev tso tawm cov enzymatic sai thiab ceev cov ntawv sau ntawm N-glycans.Cov txheej txheem no tau lees paub siv cov tshuaj tiv thaiv kab mob monoclonal thiab kuj tau sim ua rau ntau yam ntawm lwm cov N-txuas glycoproteins.Peb pom zoo cov neeg siv kom paub meej tias qhov tso tawm enzymatic rau lawv cov qauv tshwj xeeb.
Cheebtsam
N-Cov khoom siv Glycan (24T)
| Module | Component | HCP0031A |
|
Deglycosylation Module HCP0031A-1 | IgG | 1.2 mg ib |
| PNGase F | 45 l ;ua | |
| PNGase F tsis | 0.5 ml yog ' | |
| Surfactant | 1.2 mg x 6 | |
| Labeling Module HCP0031A-2 | MS reagent hmoov | 8.2mg * 3 |
| Tsis muaj dej DMF | 1 mL ib | |
| Ntxuav Module HCP0031A-3 | Elution Phaj (96well) | 1 qob |
| Elution Buffer | 5ml * 1 | |
| Qauv Diluent | 5ml * 1 | |
|
Qauv Sau Module HCP0031A-4 | 8-tube strips (200μL) | 100 daim |
| 8-cap strips | 100 daim | |
| Cov ntawv sau (96well) | 1 qob | |
| khib nyiab | 1 qob |
raws tu qauv
Tus neeg siv yuav tsum tau npaj cov khoom siv hauv qab no thiab cov khoom siv ua ntej koj pib (Ib qho qauv IgG piv txwv):
(1) 0.85% Nacl, 100µL;acetonitrile (LC-MS qib); dej ultrapurure;
(2) 15/85 = ultrapurure dej / acetonitrile tov (v/v), 2mL;1/9/90 = formic acid/ultrapure water/acetonitrile(v/v), 20mL; 50 mM ammonium formate, PH = 4.4,500mL;
(3) SPE lub tshuab nqus tsev twj;96-zoo-phaj siab processor lossis zoo siab processor;Cua sov module lossis hlau da dej (90 ℃ thiab 50 ℃);vortex tov khoom; pipette.
Kauj Ruam 1: Rapid Deglycosylation
(1) Dilute IgG qauv nrog 0.85% Nacl kom tau txais IgG kawg concentration ntawm 2mg / mL.
(2) PNGase F tsis muaj: dilute 1 vial surfactant (1.2mg / 3.6mg) nrog (24μL / 72μL)) PNGase F tsis, sib tov thiab khaws cia ntawm chav tsev kub (cov tshuaj no yuav tsum tau npaj ua ntej siv).
(3) Ntxiv 20μL IgG tov rau 200μL cov tshuaj tiv thaiv raj, ntxiv 3μL PNGase F tsis, pipette nce thiab nqis los sib tov thiab tom qab ntawd ntxiv 3.3μL ultrapure dej, aspirate thiab dispense mus sib tov.
(4) Ntxiv tag nrho cov saum toj no tov rau cua sov module ntawm 90 ℃, 3 feeb, denature nws, thiab ces muab nws tawm ntawm lub cua sov module, thiab txias rau 3 feeb ntawm chav tsev kub.
(5) Ntxiv 1.2μL PNGase F, aspirate thiab dispense rau sib tov, cua sov 50 ℃, 5 feeb nrog cua sov module.Tus neeg siv tuaj yeem npaj daim ntawv lo reagent thaum tos.
(6) Tshem tawm ntawm lub tshuab cua sov, thiab tom qab ntawd txias rau 3 feeb ntawm chav tsev kub.
Kauj Ruam 2. Kev Sau Npe nrawm ntawm Glycosylamines
(1) Labeling reagent tov: dilute ib vial labeling reagent (8.2mg / 24.6m) nrog anhydrous DMF (60. 13μL / 180.4μL), cov tshuaj no yuav tsum tau npaj ua ntej siv), aspirate thiab dispense 5- 10 zaug kom paub meej cov reagent yog yaj tag nrho.
(2) Ntxiv 6μL labeling reagent tov rau cov tshuaj tiv thaiv tube, aspirate thiab dispense 5- 10 zaug kom ntseeg tau tias sib tov, thiab tom qab ntawd tso cai rau cov tshuaj tiv thaiv kab mob mus rau hauv chav tsev.
kub rau 5 feeb.
(3) Ntxiv 179 μL acetonitrile, sib tov rau cov kauj ruam tom ntej.
Kauj ruam 3: Clean-up ntawm Lub npe Glycosylacov mines
(1) Teeb tsa lub siab tsis zoo lossis lub siab zoo processor thiab 96-zoo elution phaj.
(2) Cov qhov dej txias: ntxiv 200μL ultrapure dej rau lub phaj elution kom kho cov qhov dej, sau cov khib nyiab los ntawm cov khib nyiab.
(3) Sib npaug qhov dej: ntxiv 200μL 15/85 dej / acetonitrile tov (v/v) kom sib npaug ntawm qhov dej, sau cov khib nyiab los ntawm cov khib nyiab pov tseg.
(4) Cov qauv thauj khoom: thauj cov acetonitrile diluted samples thiab sau cov khib nyiab los ntawm cov khib nyiab pov tseg.
(5) Ntxuav lub qhov dej nrog 600μL 1/9/90 formic acid/ultrapure water/acetonitrile(v/v) ob zaug thiab sau cov khib nyiab pov tseg.
(6) Tshem tawm lub tais pov tseg, thiab hloov nws mus rau lub tais ntim khoom.
(7) Ntxuav lub cell nrog 50μL elution tsis, elute glycans rau lub phaj sau, thiab rov ua cov kauj ruam no.
(8) Ntxiv 200μL qauv diluent, sib tov nws.
Kauj ruam 4. HILIC-FLR
(1) Chromatography kem: ACQUITY UPLC® Glycan BEH Amide, 130 Å, 1.7 μm, 2. 1 x 150 mm (dej qhov # 186004742).
(2) Kub ntawm kab: 60 ° C
(3) Ua kua theem A: 50 mM ammonium formate tov (LC-MS qib tau pom zoo), pH = 4.4.
(4) Ua kua theem B: 100% acetonitrile (LC-MS qib yogpom zoo).
(5) Tus nqi ntws: 0.4 mL / min;
(6) Gradient:
| Sijhawm (min) | Tus nqi ntws (mL / min) | %A | %B | Curve |
| 0 | 0.4 | 25 | 75 | 6 |
| 35 | 0.4 | 46 | 54 | 6 |
| 36.5 ib | 0.2 | 100 | 0 | 6 |
| 39.5 ib | 0.2 | 100 | 0 | 6 |
| 43.1 ib | 0.2 | 25 | 75 | 6 |
| 47.6 ib | 0.4 | 25 | 75 | 6 |
| 55 | 0.4 | 25 | 75 | 6 |
(7) FLR wavelengths: EX 265 / EM 425 nm
(8) FLR sampling tus nqi: 2 Hz
(9) Txhaj tshuaj vol.: 10μL
Thov nco ntsoov tias cov kev ntsuas saum toj no raws li lub tshuab nruab nrog "ACQUITY® RDa" LCMS, tus neeg siv tuaj yeem kho qhov ntsuas raws li lwm yam khoom siv.
Storage
| Reagent | Cia |
| IgG | 2-8 ℃, 12 lub hlis |
| PNGase F | -25 ～ -15 ℃, 12 lub hlis, zam khov/thaw cycles |
| PNGase F tsis | 2-8 ℃, 12 lub hlis |
| Surfactant | Chav ntsuas kub, 12 lub hlis |
| MS reagent hmoov | 2-8 ℃, 12 lub hlis, khaws cia hauv qhov tsaus ntuj |
| Tsis muaj dej DMF | Chav tsev kub, 12 lub hlis |
| Elution Buffer | Chav tsev kub, 12 lub hlis |
| Qauv diluent | Chav tsev kub, 12 lub hlis |
| 96 zoo elution phaj | Chav tsev kub, ib zaug qhib, lub qhov dej tsis siv yuav tsum tau ntim nrog cov yas paraffin zaj duab xis thiab khaws cov desiccant ntawm chav tsev kub. |
| 200μL strips ntawm 8 raj, strips ntawm 8 lub kaus mom, 96 qhov chaw khaws cov phaj, khib nyiab | Chav tsev kub, 96 zoo sau phaj yuav raug kaw thiab khaws cov desiccant ntawm chav tsev kub |
Sau ntawv
Rau koj txoj kev nyab xeeb thiab kev noj qab haus huv, thov hnav lub tsho (lab) thiab hnab looj tes.
Siv cov lus qhia huv txhua lub sijhawm
Thov ua raws li phau ntawv qhia kom khaws thiab siv cov tshuaj reagents
