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1、WESTERN BLOT ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 1.5 1.0 X 40ul? 50ul?30ul? 20ul? ?10% Ddh2O4.0Ml 30%Acr3.3Ml 1.5M Tris?Ph8.8?2.5Ml 10%Sds 100Ul 10%Aps100Ul Temed4Ul ?10Ml ? DdH2O3.4Ml 30%Acr830Ul 1M Tris?Ph6.8?630Ul 10%Sds50Ul 10%Aps50Ul Temed5Ul ? 5Ml 4? -20? 4? 4? -20? 4? ? +?
2、 ? ? ? 4? ? ? TEMED ? ? ? ? ? ? 4? ? ? ? ? ? ?1.5cm? ? ? ?1.5cm?ddH2O? ?20min? ? ? 200-1000ul10-200ul0.1-10ul ? ? ? ? ? ? ? ? ?Marker? ?OD? ? ?Marker?2ul Marker ? -20? ? 5SDS-PAGE? tris 15.1g Glycine 94g SDS 5g 800ml ddH2O ? ?1L ? ?1 ? ? ? ? ?+? ?+? ? ?80V?Pr? ? ?120V? ? ?180V? ? ?1h-1.5h ? ? 5? tri
3、s 58g Glycine 29g SDS 3.7g 1200ml ddH2O ? ?1600ml ? ? ddH2O?5?3?1?1 120ml?40ml?40ml ? ?Marker? ? Smad2/355-60kDa Actin43kDa LC3B16-18kDa IL-6 21kDa COL3A1110-140kDa ? PVDF? ? ? ? ? ? PVDF?2? ? ? ? ? PVDF? ? ? ? ? Marker? ? ? ? ? ? ? 100mA 1h ?15V ? Western Blot ? ? ? TBS buffer 10 tris 24.2g NaCl 88
4、g 800ml ddH2O ? ?1L ? TBST: TBS 10?1 +Tween20?0.05%? ?500ml TBS 10 50ml?ddH2O 450ml?Tween20 250ul ? 5% ?TBST?0.5g?10ml ?V? 3% ?TBST?0.3g?10ml ? ? ? ? ? ?1h ? ?V ? ?4? ? ? ? ? Smad2/31?1500 Lc3B1?3000 Il-61?2000 Col3A11?800 Actin1?500 4?-20? ?10ml Smad2/3? ?10ml ?1015000.0067ml?6.7ul ?1h+4?+?1h ? ? ?
5、 ? ?TBST? ?+NaN3?0.03%? 0.3gNaN3?1ml ddH2O?NaN3? ?1ml?1ul NaN3? ?5min+15min+10min+10min?+10min? ? ? ? ? ? ? ?Smad2/3? ? ? ?1?3000 ?1?5000 ?1h -20? ? ? ?TBST? ?5min+15min+10min+10min?+10min? ? ECL? ? A?B?1?1? 4? ? PVDF? ?ECL? 500ul? ? ? ? ? ? ? ? ? ? Smad2/330Min Lc3B2-3Min Il-625Min Col3A130Min Actin10-15Min ? ? ? ? ? ? ? ?4-5min? ?2-3? ? PVDF?5min? ? ?
