Zogulitsa

Kuyesa kwatsopano kwamadzimadzi kwa chromatography kwa glycosyltransferase kutengera kutulutsa kwa anthranilic acid ndi kuzindikira kwa fluorescence.

Mayesero adapangidwa pogwiritsa ntchito chemistry yapadera ya 2-aminobenzoic acid (2AA; anthranilic acid, AA) poyezera zochita za β1-4 galactosyltransferase (GalT-1) ndi α2-6 sialyltransferase (ST-6) ndi madzi ochita bwino kwambiri. chromatography (HPLC) yokhala ndi fluorescence kuzindikira (Anumula KR. 2006. Kupita patsogolo kwa fluorescence derivatization njira kwa mkulu-ntchito madzi chromatographic kusanthula glycoprotein chakudya. Anal Biochem. 350: 1-23).N-Acetylglucosamine (GlcNAc) ndi N-acetyllactosamine ankagwiritsidwa ntchito monga ovomerezeka ndi uridine diphosphate (UDP) -galactose ndi cytidine monophosphate (CMP) -N-acetylneuraminic acid (NANA) monga opereka GalT-1 ndi ST-6, motero.Zogulitsa za enzymatic zidalembedwa mu situ ndi AA ndipo zidalekanitsidwa ndi magawo pagawo la TSKgel Amide 80 pogwiritsa ntchito mikhalidwe yabwinobwino.Magawo a enzyme adatsimikiziridwa kuchokera kumadera okwera kwambiri poyerekeza ndi miyezo yochokera ku Gal-β1-4GlcNAc ndi NANA-α2-6 Gal-β1-4GlcNAc.Linearity (nthawi ndi kuchuluka kwa ma enzyme), kulondola (intra- ndi interassay) ndi kubwezeredwa kwa zoyeserera zidakhazikitsidwa.Mayeserowa adapezeka kuti ndi othandiza pakuwunika ntchito za enzyme panthawi yodzipatula komanso kuyeretsedwa.Zoyesazo zinali zokhudzidwa kwambiri ndipo zinkachitidwa mofanana kapena bwino kuposa miyeso yachizolowezi yochokera ku shuga wa radioactive.Mtundu woyeserera ungagwiritsidwenso ntchito kuyeza zomwe zimachitika pakusamutsa kwina, malinga ngati olandila ma carbohydrate ali ndi malire ochepetsera zilembo.Kuyesa kwa olandila glycoprotein kudapangidwa pogwiritsa ntchito IgG.Njira yachidule ya mbiri ya HPLC idapangidwa kuti ikhale yolekanitsa ma IgG glycans (biantennary G0, G1, G2, mono- ndi disialylated), zomwe zidathandizira kutsimikizika kwa ntchito za GalT-1 ndi ST-6 mwachangu.Kuphatikiza apo, njira yowonera mbiriyi iyenera kukhala yothandiza pakuwunika kusintha kwa IgG glycans m'malo azachipatala.