- galactosidaza tir hat cd boat tinh lectin Lectin nay tuang tac dac hieu vd
p rote/>i C /^ 5/ w I)
2. 2. 3. 2. Xac dinh ham lugng protein trong man thi nghiem.
Tir đ thj nay dura vao mat đ quang OD do dugc lir cac mau thf
^ C . V . B ^(mg) " 1000
Trong đ y la sd mg protein cd trong mau
C la ndng do protein (mg/inl) tfnh theo đ Ihị V la the tfch dung djch mau thf nghiem
B la do pha loang dung djch mhị
2. 2. 4. Phuang phap xac dinh hoat do lectin.
Hoat do lectin cdn dugc ggi la hoat do ngimg kel hong cau, viet lai la HAA (Haemagglutinaling Activity). Hoat đ nay dugc xac djnh theo phuang phap Alecxandcr Ạ Kroll [721.
Mdt dan vj hoat do lectin dugc tfnh la gia In nghjch dao cua do pha loang Idn nhát cua ché pham lectin ma d đ van cdn kha nang gay ngirng kel hong caụ Dan vj nay dugc ggi la dan vj gay ngung kel hdng caụ Don vi HAA be chírng Id hoat do lectin yeụ Dan vi HAA cd gia In Idn, lectin cd hoat do manh. Cd 2 chi sd xac djnh hoal do leclin.
2. 2. 4. L Hoat do tong sd ciia lectin (HDTS)
Hoat do long sd dugc tfnh theo cdng Ihiic :
HDTS V.l"^
(d.v. HAA) 5 o y
Trong đ : V la the tfch mau thf nghiem tfnh bang |,il. n la sd giéng cdn hoat tfnh leclin
2. 2. 4. 2. Boat do rieng (HDR).
HDR. ™ T «
Xd.v HAA)/mg protein) ^ng protein
Hoal do rieng la gia tri hoat đ cua leclin trong 1 mg protein. HDR cd gia trj Idn chiitig id ly le lectin trong 1 mg protein la Idn. Gia In nay dai cue dai khi 1 mg protein chfnh la 1 mg lectin. Vi vay HDR la chi sd quan trong danh gia mirc do linh sach cua leclin irong qua irinh linh ché.
2. 2. 5. Tinh che lectin.
Chung tdi da sir dung cac hinh thiic khac nhau de linh ché leclin. Cac loai sac ky dugc sir dung la : Sac ky loc gel Sephadex, sac ky trao đi ion va sac ky ai luc.
2. 2. 5. Ị Sac ky loc gel Sephadex G - 75 va CJ - 100.
Day la phutmg phap loc sang phan lir. Dung djch de hda nil leclin la dem PBS pH 7,4. Qua trinh sac ky dugc thuc hien Iheo tCrng phan doan, mdi phan doan Ihu 3 ml. Tdc do đng chay khoang 30 ml / gid. Moi thao lac deu dugc thirc hien chat che theo yeu can cua ky thuat sac ky col.
2. 2. 5. 2. Sac ky trao đi ion DEAE - Xenlulo/a va DEAl] Trysacryl.
Qua trinh sac ky nay du Iren nguyen tac trao đi ion. Dung djch hda rut lectin la dem phot phat Nalri 0^ 2 M d pH 8,0. Qua trinh sac ky theo gradient ndng do NaCl. Mdi phan doan thu 2 ml, tdc do đng chay 20 ml/ gid.
2. 2. 5. 3. S^c ky ai luc.
Phir(tng phap nay cd hieu qua rat caọ Tuy vay nd chi dugc ap dung vdi cac dang lectin cd kha nang tuang lac dac hieu vdi mgl loai dudng nao đ va lai cd kha nang lien két dac hieu vdi mgl loai hgp chat nao do nhu Sephadex G-50 hoac Muxin. Trong qua trinh tinh che leclin, chiing Idi da
sir dung cdt ai luc Sephadex G-50. Col cd dudng kfnh 2,0 cm va chieu cao cot la 60cm. Chiing tdi da dung D-glucoza 0,2 M de day lectin bam d cot. Mdi phan doan thu 5 ml. Tdc đ đng chay 20 ml/gid. Chiing tdi da diing 2 sa do sau de tien hanh tinh ché lectin:
SO DO 1: DUNG 2 GIAI DOAN SAC KY NOI TIEP NHAỤ (Theo tac gia Do Ngoc Lien va cong su)
• i f f i f .NGUYEN Llfill
Chiet nit bang dem PBS pH 7,4 Li tam 5000 vdng/phut trong 30 phiit
ni( FumnT
Ket tua bang (NH4)2S04 60% bao hoa Li tam, lay can tua
< HEPHAMLJ:(UN run
Sac ky loc gel Sephadex G-75 va G-lOO
( H E PHAM LECTIN S<)( HK
Sac ky trao đi ion DEAE-Xenluloza va DEAE - Trysacryl
SO DO 2: TINH CHE LECTIN BANG SAC K f AI LlTC (Theo Goldstein va cong su 1986, co cai tien d budc ket tua djch
chiet bang (NH4)2S04 60% bao hoa trudc khi sac ky).