Smolke:Protocols/Western: Difference between revisions
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==Materials== | ==Materials== | ||
*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) | *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) | *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) | *MOPS running buffer (Invitrogen NP0001) - above gel bench (use MES instead for small proteins) | ||
*Nitrocellulose membrane | *Nitrocellulose membrane - in drawer below gel bench | ||
*Blotting pads | *Blotting pads - in drawer below gel bench | ||
*Transfer buffer (Invitrogen NP0006-1) | *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) | *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]) | *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 | *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 | *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 | ||
==Procedure== | ==Procedure== | ||
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#Reweigh tubes, resuspend at 100 uL/mg in 0.1 M NaOH | #Reweigh tubes, resuspend at 100 uL/mg in 0.1 M NaOH | ||
#Incubate at RT for 5 min | #Incubate at RT for 5 min | ||
#Repellet cells, resuspend at 25 uL/mg in 1x Laemmli buffer and transfer 50uL to a PCR tube | #Repellet cells, resuspend at 25 uL/mg in 1x Laemmli buffer (or use LDS buffer instead) and transfer 50uL to a PCR tube | ||
#Heat at 95 °C for 3 min | #Heat at 95 °C for 3 min (70 °C for 10 m for membrane-bound proteins) | ||
#Repellet, max speed, RT, 5 min | #Repellet, max speed, RT, 5 min | ||
#Remove supernatant | #Remove supernatant | ||
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#Take 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid) | #Take 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid) | ||
#Prep pre-cast SDS-PAGE gel | #Prep pre-cast SDS-PAGE gel | ||
#Load 20uL of each sample | #Load 20uL of each sample (20-40 ug total protein) | ||
#Run gel with MOPS running buffer, 150V, ~1hr | #Run gel with MOPS running buffer, 150V, ~1hr | ||
#*Run until dye front is near bottom of gel | #*Run until dye front is near bottom of gel | ||
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#Cut out membrane slightly larger than gel | #Cut out membrane slightly larger than gel | ||
#*Be careful not to touch membrane - use forceps. | #*Be careful not to touch membrane - use forceps. | ||
#Make 2x transfer buffer + 10% MeOH | #Make 2x transfer buffer + 10% MeOH (use 20% for small proteins, or use none and use PVDF membrane for large proteins) | ||
#Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes | #Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes | ||
#Soak pads + membrane in 2x transfer buffer + 10% MeOH | #Soak pads + membrane in 2x transfer buffer + 10% MeOH |
Latest revision as of 14:18, 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 |