Jhdavis::Western::Eyal: Difference between revisions
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(New page: 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 Pro...) |
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==running the western== | |||
soak gel in cold 1x transfer 5 min, wet PVDF membrane in MeOH then soak along with blotting paper in 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): | 10x PBS (1L): | ||
20 g NaCl (344 mM) | 20 g NaCl (344 mM) | ||
Revision as of 09:55, 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 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
