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N-Glycan Kit HCP0031A Kiʻi Manaʻo
  • N-Glycan Kit HCP0031A

N-Glycan Kit


Helu popoki: HCP0031A

Pūʻolo: 5T / 24T / 96T

Hāʻawi kēia palapala i ka ʻike e pili ana i ka mālama maʻamau a me ka hoʻohana ʻana i ka N-Glycan Kit no ka hoʻokuʻu ʻana i ka enzymatic wikiwiki a me ka lepili wikiwiki o N-glycans.

Hōʻike huahana

ʻikepili huahana

Hōʻike huahana

Hāʻawi kēia palapala i ka ʻike e pili ana i ka mālama maʻamau a me ka hoʻohana ʻana i ka N-Glycan Kit no ka hoʻokuʻu ʻana i ka enzymatic wikiwiki a me ka lepili wikiwiki o N-glycans.Hoʻopaʻa ʻia kēia protocol me ka hoʻohana ʻana i nā antibodies monoclonal a ua hoʻāʻo ʻia e hana no kahi ākea o nā glycoproteins pili N-pili.Paipai mākou i ka mea hoʻohana e hōʻoia i ka hoʻokuʻu enzymatic no kā lākou laʻana.


  • Mua:
  • Aʻe:

  • Nā ʻāpana

    N-Kiko Glycan(24T)

    Module

    Cmea hoʻokūkū

    HCP0031A

     

    Deglycosylation Module

    HCP0031A-1

    IgG

    1.2 mg

    PNGase F

    45µL

    PNGase F pale

    0.5mL

    Mea hoʻokuʻu

    1.2mg*6

    Lepili Module

    HCP0031A-2

    MS reagent pauda

    8.2mg*3

    DMF anhydrous

    1mL

    Hoʻomaʻemaʻe Module

    HCP0031A-3

    Papa Elution (96well)

    1 apana

    Elution Buffer

    5mL* 1

    Laʻana Diluent

    5mL* 1

     

    Laʻana Hōʻiliʻili Module

    HCP0031A-4

    Nā ʻāpana 8-paipu (200μL)

    100 mau ʻāpana

    8-kape

    100 mau ʻāpana

    Papa ohi(96well)

    1 apana

    pā ʻōpala

    1 apana

    Kūkākūkā

    Pono ka mea hoʻohana e hoʻomākaukau ma lalo o nā reagents a me nā mea hana ma mua o kou hoʻomaka ʻana (Hoʻokahi laʻana IgG ma ke ʻano he laʻana):

    (1) 0.85% Nacl, 100µL;acetonitrile( LC-MS māka);ka wai ultrature;

    (2)15/85=ka wai ultrature/acetonitrile solution (v/v), 2mL;1/9/90=ʻakika formic/wai ultrature/acetonitrile(v/v),20mL;50 mM ammonium formate,PH=4.4 ,500mL;

    (3) SPE ʻūmaʻu pauma;96-well-plate kaomi ʻino ʻino a i ʻole kaʻina hana kaomi maikaʻi;Module hoʻomehana a ʻauʻau metala (90 ℃ a me 50 ℃);mea hui vortex;pipette.

     

    KaʻAnuʻu Hana 1: Deglycosylation wikiwiki

    (1) E hoʻoheheʻe i ka laʻana IgG me 0.85% Nacl e loaʻa ai ka IgG hoʻopaʻa hope ma 2mg/mL.

    (2) PNGase F buffer: dilute 1 vial surfactant (1.2mg/3.6mg) me (24μL/72μL)) PNGase F buffer, hui a mālama i ka lumi wela (pono e hoʻomākaukau i kēia hopena ma mua o ka hoʻohana ʻana).

    (3) E hoʻohui i ka 20μL IgG solution i ka 200μL reaction tube, e hoʻohui i ka 3μL PNGase F buffer , pipette i luna a i lalo e hui ai a laila hoʻohui i ka wai ultrapure 3.3μL, aspirate a hoʻokuʻu e hui.

    (4) E hoʻohui i nā mea hoʻonā a pau i luna i ka module hoʻomehana ma 90 ℃, 3 mau minuke, denature ia, a laila e hoʻokuʻu iā ia mai ka module hoʻomehana, a hoʻomaha no 3 mau minuke i ka mahana wela.

    (5) Hoʻohui i ka 1.2μL PNGase F, aspirate a hoʻokuʻu e hui, wela 50 ℃, 5 mau minuke me ka module hoʻomehana.Hiki i ka mea hoʻohana ke hoʻomākaukau i ka lepili reagent oiai e kali ana.

    (6) E hoʻokuʻu iā ia mai ka module hoʻomehana, a laila e hoʻomaha no 3 mau minuke ma ke ana wela.

     

    KaʻAnuʻu 2. Lepili wikiwiki o Glycosylamines

    (1) Lepili reagent solution: e hoʻoheheʻe i hoʻokahi vial labeling reagent (8.2mg/24.6m) me DMF anhydrous (60. 13μL/180.4μL), pono e hoʻomākaukau i kēia hopena ma mua o ka hoʻohana ʻana), aspirate a hāʻawi i nā manawa 5-10 e hōʻoia ai. ua hoʻoheheʻe ʻia ka reagent.

    (2) Hoʻohui i ka 6μL labeling reagent solution i ka paipu hopena, aspirate a hāʻawi i nā manawa 5-10 e hōʻoia i ka hui ʻana, a laila e ʻae i ka hopena lepili e hoʻomau i ka lumi.

    wela no 5 minuke.

    (3) Hoʻohui i ka 179 μL acetonitrile, hui no ka pae aʻe.

     

    KaʻAnuʻu 3: Hoʻomaʻemaʻe i ka Kapa ʻia ʻo Glycosylanā mines

    (1) E hoʻonohonoho i ka mea hoʻoheheʻe ʻino ʻole a i ʻole ka ʻōnaehana kaomi maikaʻi a me ka pā elution 96-well.

    (2) Nā pūnāwai kūlana: hoʻohui i ka wai ultrapure 200μL i ka pā elution e hoʻomaʻamaʻa i nā pūnāwai, e hōʻiliʻili i ka ʻōpala ma ka pā ʻōpala.

    (3)Hoʻoponopono i nā pūnāwai: hoʻohui i 200μL 15/85 wai/acetonitrile solution (v/v) e hoʻohālikelike i nā pūnāwai, e hōʻiliʻili i ka ʻōpala ma ka pā ʻōpala.

    (4) Hoʻouka i ka laʻana: E hoʻouka i ka acetonitrile i hoʻoheheʻe ʻia nā laʻana a hōʻiliʻili i ka ʻōpala ma ka pā ʻōpala.

    (5) Holoi i nā pūnāwai me 600μL 1/9/90 formic acid/ka wai ultrature/acetonitrile(v/v) ʻelua a hōʻiliʻili i ka ʻōpala ma ka pā ʻōpala.

    (6) Wehe i ka pā ʻōpala, a hoʻololi i ka pā hōʻiliʻili.

    (7) Holoi i ke kelepona me 50μL elution buffer, elute glycans i ka pā hōʻiliʻili, a hana hou i kēia kaʻina.

    (8) Pākuʻi 200μL hāpana diluent, hui ia.

    KaʻAnuʻu 4. HILIC-FLR

    (1) kolamu Chromatography: ACQUITY UPLC® Glycan BEH Amide, 130 Å, 1.7 μm, 2. 1 x 150 mm(wahi wai #186004742).

    (2) Ka wela o ke kolamu: 60 °C

    (3) Māhele wai A:50 mM ammonium formate solution ( LC-MS grade is well as well as LC-MS grade ), pH=4.4.

    (4) Māhele wai B: 100% acetonitrile (ʻo ka papa LC-MSpaipai ʻia).

    (5) Kahe kahe: 0.4 mL/min;

    (6) Gradient:

    Manawa(min)

    Laki kahe (mL/min)

    %A

    %B

    Kipi

    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)Nā nalu FLR: EX 265/EM 425 nm

    (8)FLR sampling rate: 2 Hz

    (9) Injection vol.: 10μL

    E ʻoluʻolu e hoʻomaopopo i nā ʻāpana ma luna e pili ana i ka mīkini i hoʻolako ʻia me "ACQUITY® RDa" LCMS, hiki i ka mea hoʻohana ke hoʻoponopono i ka ʻāpana e like me nā lako ʻē aʻe.

     

    Shoʻomāʻewaʻewa

    Reagent

    Waihona

    IgG

    2-8 ℃, 12 mahina

    PNGase F

    -25 ~ – 15 ℃ , 12 mahina , pale

    nā pōʻai maloʻo maloʻo

    PNGase F pale

    2-8 ℃, 12 mahina

    Mea hoʻokuʻu

    Ka Mahana lumi, 12 mahina

    MS reagent pauda

    2-8 ℃, 12 mahina, mālama i ka pōʻeleʻele

    DMF anhydrous

    Ka wela lumi, 12 mahina

    Elution Buffer

    Ka wela lumi, 12 mahina

    Laʻana diluent

    Ka wela lumi, 12 mahina

    96 well elution plate

    ʻO ka mahana o ka lumi, i ka wā i wehe ʻia ai, pono e hoʻopaʻa ʻia ka punawai i hoʻohana ʻole ʻia me ka kiʻi paraffin plastic a mālama i ka desiccant i ka lumi wela.

    200μL paʻi o 8 paipu, paʻi o

    8 kapole, 96 punawai papa ohi, pa'i 'ōpala

    Ka wela lumi, 96 luawai ohi

    e hoʻopaʻa ʻia ka pā a mālama i ka desiccant ma ka lumi wela

     

    Nā memo

    No kou palekana a me kou olakino, e ʻoluʻolu e hoʻokomo i ka lole lab a me nā mīkina lima.

    E hoʻohana i nā ʻōlelo aʻoaʻo maʻemaʻe i kēlā me kēia manawa

    E ʻoluʻolu e hahai i ka manual e mālama a hoʻohana i nā reagents

     

    E kākau i kāu leka ma aneʻi a hoʻouna mai iā mākou