Smolke:Protocols/Western: Difference between revisions
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*Y-PER ([http://www.piercenet.com/products/browse.cfm?fldID=06010430 Pierce]) | *Y-PER ([http://www.piercenet.com/products/browse.cfm?fldID=06010430 Pierce]) | ||
*Halt EDTA-free Protease Inhibitor ([http://www.piercenet.com/Products/Browse.cfm?fldID=02040806 Pierce]) | *Halt EDTA-free Protease Inhibitor ([http://www.piercenet.com/Products/Browse.cfm?fldID=02040806 Pierce]) | ||
*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) | |||
*Prestained protein ladder (Invitrogen LC5800) | |||
*Protein loading buffer | |||
*MOPS buffer (Invitrogen NP0001) | |||
*Nitrocellulose membrane | *Nitrocellulose membrane | ||
*Blotting pads | |||
*Transfer buffer (Invitrogen NP0006-1) | |||
*Methanol | |||
*Anti-V5-HRP antibody (Invitrogen R961-25) | |||
*Chemiluminescence detection kit ([http://www.piercenet.com/Products/Browse.cfm?fldID=01041101 Pierce]) | |||
==Procedure== | ==Procedure== | ||
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#Run gel with MOPS running buffer, 150V, ~1hr | #Run gel with MOPS running buffer, 150V, ~1hr | ||
===Transfer=== | ===Semi-dry Transfer=== | ||
#Make 2x transfer buffer + 10% MeOH | #Make 2x transfer buffer + 10% MeOH | ||
#Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes | #Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes |
Revision as of 15:11, 23 January 2009
Still a work in progress OverviewBlotting for V5-tagged proteins in S. cerevisiae Materials
ProcedureLysis
SDS-PAGE
Semi-dry Transfer
Blotting
NotesReferencesContact |