Jhdavis::Western::Eyal: Difference between revisions
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==running the western== | ==running the western== | ||
soak gel in cold 1x transfer 5 min, wet PVDF membrane in MeOH then soak along with blotting paper in transfer | soak gel in cold 1x transfer 5 min, wet PVDF membrane in MeOH then soak along with blotting paper in 1x transfer<br> | ||
assemble: | assemble:<br> | ||
--anode (red)-- | --anode (red)--<br> | ||
--blotting paper-- | --blotting paper--<br> | ||
--blotting paper-- | --blotting paper--<br> | ||
--PVDF-- | --PVDF--<br> | ||
--gel-- | --gel--<br> | ||
--blotting paper-- | --blotting paper--<br> | ||
--blotting paper-- | --blotting paper--<br> | ||
--cathode (black)-- | --cathode (black)--<br> | ||
transfer at 1mA/cm2 for 1 hour | transfer at 1mA/cm2 for 1 hour | ||
Revision as of 09:56, 15 May 2008
running the western
soak gel in cold 1x transfer 5 min, wet PVDF membrane in MeOH then soak along with blotting paper in 1x transfer
assemble:
--anode (red)--
--blotting paper--
--blotting paper--
--PVDF--
--gel--
--blotting paper--
--blotting paper--
--cathode (black)--
transfer at 1mA/cm2 for 1 hour
10x Transfer Buffer: 250 mM Tris 1.92 M Glycine mixture pHs itself to pH 8.5
1x Transfer Buff: 80 mL 10x 720 mL H20 200 mL Methanol
10x PBS (1L): 20 g NaCl (344 mM) 2 g KCl (27 mM) 14 g Na2HPO4 2 g KH2PO4
1x PBS-tween: 1x PBS Tween20 at 2 mL/L
Blocking Buffer: 5% Milk 0.02% Na-Azide PBS-Tween
Washing: PBS-Tween
Protocol: 1 hour block add primary 1:10,000 o/n 3x wash 1 hour with secondary 1:10,000 4x wash ECL exposure
