Jhdavis::Western::Eyal: Difference between revisions
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transfer at 1mA/cm2 for 1 hour | transfer at 1mA/cm2 for 1 hour | ||
<nowiki> | |||
10x Transfer Buffer: | 10x Transfer Buffer: | ||
250 mM Tris | 250 mM Tris | ||
1.92 M Glycine | 1.92 M Glycine | ||
mixture pHs itself to pH 8.5 | mixture pHs itself to pH 8.5 | ||
</nowiki> | |||
1x Transfer Buff: | 1x Transfer Buff: | ||
80 mL 10x | 80 mL 10x | ||
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
