:
e , -, (Oxoid, ), QIAGEN-tip 500-Max (QIAGEN, ͳ); Effectene Transfection Reagent (QIAGEN, ͳ); DMEM, (FBS), , L-, , - Versene ( - Gibco, Life Technologies, ); Any kD MiniPROTEAN TGX Gel; - (BioRad, ); Pierce Unstained Protein Molecular Weight Marker (ThermoScientific, ); TransBlot Turbo Nitrocellulose Transfer Packs (BioRad, ); SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific, ); , Triton X100, Tween 20%, , -, ( - SigmaAldrich, ), .
: AntiFLAG M2 (SigmaAldrich, ); AntiMouse HRP, AntiRabbit IgG (Chemicon, ); , - AntiR3 CHO_3pur, IgG MTS-T 14/04/85, IgG1 Gln-DEAE 14/04/85, IgG anti cKRS DN lapin 117 25/04/02, IgG AspRS DN20 13/12/96, IgG anti human p43 Ct lapin 052 30/05/00.
.
: Scepter 2.0 Cell Counter (Millipore); 2- MCO20AIC (Sanyo, ); Heraeus Pico 17 Heraeus Fresco 21 (ThermoScientific, ), a Beckman J20 (Beckman, ); Gel Doc EZ System (BioRad, ); Trans Blot Turbo Transfer Starter System (BioRad); Western-blot Bench-Pro 4100 Card Processing Station (Invitrogen); Luminescent Image Analyzer LAS3000 (Fujifilm Life Science); ; Varian 50 (Varian, ).
pc5FLAG/TUSC4 ' ﳿ (, ) , pCMV6-XL5/MRS, pCMV6-XL5/QRS, pCMV6-XL5/KRS, pCMV6-XL5/DRS, pCMV6-XL5/p43 , .
HeLa HC , () . DMEM 10% , 2 L- (50 /) (50 /) 5% 2, 100% 370.
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|
3.3. ϳ
, pCMV6-XL5 150 . 370 16 0,35-0,4 OD.
QIAGEN-tip 500-Max (QIAGEN). . -200.
3.4. ϳ HeLa
HeLa 75 2 DMEM 80%. ϳ , 2 Versene 20-30 . ³ 8 .
Scepter 2.0 Cell Counter (Millipore). ϳ . ³ DMEM 0,25*106 /.
0,5*106 , 10 ( 8 , 56,7 2). 24 5% 2, 100% 370.
.