Smolke:Protocols/Western: Difference between revisions
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==Materials== | ==Materials== | ||
*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) - in cold room | *NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) - in cold room | ||
*Prestained protein ladder (NEB P7711S) - | *Prestained protein ladder (NEB P7711S) - 8 uL aliquots in freezer by LC-QQQ | ||
*Protein loading buffer (NuPage LDS sample buffer, Invitrogen NP0007) - in | *Protein loading buffer (NuPage LDS sample buffer, Invitrogen NP0007) - small bottle on shelf above gel bench, large bottle in deli fridge (let warm to room temp for LDS to redissolve, then aliquot into small bottle) | ||
*MOPS buffer (Invitrogen NP0001) - above gel bench | *MOPS running buffer (Invitrogen NP0001) - above gel bench (use MES instead for small proteins) | ||
*Nitrocellulose membrane - in drawer below gel bench | *Nitrocellulose membrane - in drawer below gel bench | ||
*Blotting pads - in drawer below gel bench | *Blotting pads - in drawer below gel bench | ||
*Transfer buffer (Invitrogen NP0006-1) - above gel bench | *Transfer buffer (Invitrogen NP0006-1) - above gel bench | ||
*Methanol | *Methanol | ||
*Anti-V5-HRP antibody (Invitrogen R961-25) - if HA-tagged, anti-HA-HRP antibody (Abcam ab1188) - aliquoted in | *Anti-V5-HRP antibody (Invitrogen R961-25) - if HA-tagged, anti-HA-HRP antibody (Abcam ab1188) - aliquoted in freezer by LC-QQQ | ||
*Chemiluminescence detection kit ([http://www.piercenet.com/Products/Browse.cfm?fldID=01041101 Pierce]) - in drawer below gel bench | *Chemiluminescence detection kit ([http://www.piercenet.com/Products/Browse.cfm?fldID=01041101 Pierce]) - in drawer below gel bench | ||
*BSA fraction V or dried milk for blocking - in cold room | *BSA fraction V or dried milk for blocking - made from BSA in deli fridge, stock stored in cold room | ||
*10x TBST solution (80 g NaCl, 30 g Tris, add ~850 mL MP water, adjust pH to 8 with concentrated HCl, add 5 mL Tween 20 (or make TBS and add tween to each 1x bottle), fill to 1 L) - in cold room | *10x TBST solution (80 g NaCl, 30 g Tris, add ~850 mL MP water, adjust pH to 8 with concentrated HCl, add 5 mL Tween 20 (or make TBS and add tween to each 1x bottle), fill to 1 L) - in cold room | ||
Revision as of 14:15, 23 August 2014
OverviewBlotting for large V5-tagged proteins in S. cerevisiae Materials
ProcedureLysis
SDS-PAGE
Semi-dry Transfer
Blotting
Notes
ReferencesKushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860. Contact |