2. 2.1.
2.1.1. : : Vortex-GENIE 2 : Sartorius CP1245-OCE : Sartorius
CP2201 : HiTrap IgM purification, 1ml, GE Healthcare, code No.
17-5110-01 : HiTrap protein G HP, 1ml, GE Healthcare, code No.
17-0404-01 : BRANSON 2200 : Stuart,snijders 34532 Heidolph unimax
1010 : HANNA : Mini-Protean II Mini-Protean III Bio-Rad :
Mini-Trans-Blot Bio-Rad : Millipore Minitan system : Julabo o /
Bio-Rad Smartspec plus : Eppendorf 581012 Cryotubes: Nunc Elisa
reader: Bio-Rad model 6802.1.2:
falcon 15ml falcon 50ml eppendorf 1,5 ml 2,5,10 & 25
ml2.1.3:
Goat anti mouse IgM / HRP, ThermoScientific Prod 31440
Goat anti-Mouse IgG/HRP, ThermoScientific Prod 31413
Goat anti- Rabbit/HRP, Dako Prod. P0448.
Rabbit anti Hepcidin25 , Bachem T-4822.0500
Rabbit anti mouse Ig/HRP, Dako Prod. #P0161.
2.1.4: Kit TMB Substrate kit (Peroxide Solution / Peroxide
Substrate), Pierce, Prod. #34021 Complete Freunds Adjuvant (CFA)
(Sigma, USA) Freund. ncomplete Freunds adjuvant (IFA) (Sigma, USA)
Freund.2.1.5.:PBS 10X, pH 7.4:
137mM NaCl
2.7mM KCl 10mM Na2HPO4 2mM KH2PO4 (separating gel) (V=10mL):
1.5M Tris-HCl, pH 8.8: 2.5mL
10% : 0.1mL
10% w/v SDS 10X: 0.1mL
TEMED (SIGMA): 0.004mL (stacking gel) (V=10mL): 30% : 1.7mL
0.5M Tris-HCl, pH 6.8: 1.25mL
10% : 0.1mL
10% w/v SDS 10: 0.1mL
TEMED: 0.01mL
(Sample buffer): 45mM Tris-HCl, pH 6.8 10% 1% SDS 10X 0.01% 5%
2-Electrode buffer 10X, pH 8.5: 25mM Tris-base, pH 8.5 200mM : 10%
electrode buffer 10 1% SDS 10
(Coomasie stain buffer:)
0.1% Coomassie R-250 40%
10%
(Destain buffer): 45% 10%
HiTrap Protein G (binding buffer): 0.02M (sodium phosphate), pH
7.0 HiTrap Protein G (elution buffer): 0.1M -Cl, pH 2.72.1.6: ,
L-1: (Human), Peptide Institute INC., code 4392-s, Lot 610310: ,
Peptide Institute . : NH2-
Asp-Thr-His-Phe-Pro-Ile-Cys-Ile-Phe-Cys-Cys-Gly-Cys-Cys-His-Arg-Ser-Lys-Cys-Gly-Met-Cys-Cys-Lys-Thr-COOH.
:1-8, 3-6, 2-4 & 5-7. Hep25-His: , (), , P. pastoris. LEAP-1, 6
, ( Ni2). :NH2-
Asp-Thr-His-Phe-Pro-Ile-Cys-Ile-Phe-Cys-Cys-Gly-Cys-Cys-His-Arg-Ser-Lys-Cys-Gly-Met-Cys-Cys-Lys-Thr-His-His-His-His-His-His-COOH.
Hep25: , (), , P. pastoris. : NH2-
Asp-Thr-His-Phe-Pro-Ile-Cys-Ile-Phe-Cys-Cys-Gly-Cys-Cys-His-Arg-Ser-Lys-Cys-Gly-Met-Cys-Cys-Lys-Thr-COOH.(
LEAP-1). KLH (keyhole lympet hemocyanin): M (~350 kD). Megathura
crenulata ( ) , . BSA (Bovine Serum Albumin): . ( .. ELISA, Western
Blot) .2.1.7. DMSO (): , -800C . FBS 1:10. FBS( Fetal Bovine
Serum): Gibco #10270
Hybridoma Fusion and Cloning Supplement (HFCS) (50x) (Roche,
USA). Hypoxanthine Aminopterin Thymidine (HAT) (50x) (Gibco, UK). .
Hypoxanthine Thymidine (HT) (50x) (Gibco, UK). L-Glutamine
solution, Sigma #G-1146, (200 mM) PenStrep
(Penicillin-Streptomycin): Gibco #15140, Penicillin(10,000
U)-Streptomycin(10 mg) RPMI 1640+ Glutamax 1: Gibco #61870
Trypan Blue Stain 0,4% (Gibco, UK) Ultra Low IgG FBS: Gibco
#16250 PEG() 1500 50%, w/v: (Roche, USA).2.2: 2.2.1: ELISA
(enzyme-linked immunosorbent assay)
ELISA, , , , . ELISA, .
2.2.1.1: . ELISA , . , :
1. PBS 1g/ml. , 50l . 12 4C.
2. PBS, pH 7.4.(1 ), .3. PBS-0,05% tween-2%BSA, 100 l . , . 1
37C.4. PBS-tween 0.05%, pH 7.4 (2 ).
5. . PBS-tween 0.05%-1%BSA 50 l . 6. , (), . 1 , 37C 7.
PBS-tween 0.05%, pH 7.4(3 ), .8. PBS-tween 0.05%-1% BSA, .
HRP(Horse Radish Peroxidase) . , 50l . 1 , 37C.9. PBS-tween 0.05%,
pH 7.4 (3 ), .
10. ELISA . 3,3',5,5'- , . TMB Substrate kit (Peroxide Solution
/ Peroxide Substrate) Pierce. (50l ) 620 nm.
11. , 1 H2SO4 (50l/ ). , , 450 nm. 620 nm .
2.2.1.2: ELISA .2.2.2: .
, . , , . , GE Healthcare. IgG protein G HP (1ml), G. G IgG , Fc
. pH, pH. .
2.2.2.1: IgG HiTrap protein G.
:1. 1200rpm, 5 , 45m. , .2. 1:3 (binding buffer, 0.02M sodium
phosphate, pH 7).
3. (HiTrap, protein G) , 2ml.4. , . 60 /. falcon (flow
through).
5. (10 ml). , G. falcon (washing).
6. (0,1 ,pH 2.7). eppendorf, 25-40 l 1 Tris, pH 9.0. eppendorf
1ml 1 Tris, pH 9.0 , pH eppendorf 7. pH . 30 /.7. eppendorf. , 280
nm IgG 1mg/ml i 1,36. IgM 1,2.
8. PBS,pH 7.4, 16 4C, . 12kDa . 9. PBS, 280 nm, -20C. 50% (: ).
, SDS-PAGE . , ELISA, .2.2.3: SDS-PAGE(PolyAcrylamide Gel
Electrophoresis) . , , . (SDS-PAGE) . SDS 2 , , SDS- , . SDS , .
2.2.3.1: .
. . , : (ammonium persulfate, APS) TEMED. , . , : (stacking gel)
(resolving gel) . Laemmli, , . , . , (running buffer) 200 V . SDS,
. 2-, , -SH . 96 C 5 .
2.2.3.1.1: 2.2.3.2: .
Coomassie Brilliant Blue ( >1g) 30 , 370C, . (Destain
buffer), 45 , 370C . .2.2.4: Balb/c & C57Black/6, 6 ( ). 2 :
(subcoutaneous, sc.) (intraperitoneal, ip.)
2.2.4.1:
:
1. C57Black/6 Hep25 (s.c., subcoutaneous), (i.p.,
intra-peritoneal). , Balb/c KLH-LEAP. , PBS, pH 7.4 Freud (FCA). 50
g ( ). . , .2. 15 , . 25g. Freud (IFA) .
3. 9-10 , ELISA . , .4. . 2.5. . 75g . 4-5 .
2.2.5: (fusion). , . :
sp2, (PEG) . , HAT, (), () (). de novo . . , , , . , 1-2 , ,
.
2.2.5.1: . :
1. , / ml Neubauer x/4**104. Trypan blue, , .2. 2 (RPMI
1640,PEN/STR), (FBS), . 800 rpm, 5 , (RT).
3. .
4. 5:1. RPMI ( FBS). 50ml 5 , 600rpm, (RT).
5. , 37C 6. 2ml (PEG 1500), 2 . - . ( 37C)7. 1ml RPMI( FBS) 2 .2
.
8. , 5 9ml RPMI ( FBS), 2-.9. 5ml RPMI ( FBS 2-) 600rpm 10 ,
(RT).10. RPMI, : PEN/STR(1x), ), 5% FBS, L-glut (1x), 2-ME (0,05),
HAT (1x), HCSF (1x). 96 , 200l/.
