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 | |||
*Prestained protein ladder (NEB P7711S) - aliquoted in ME bay freezer | |||
*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) | *Protein loading buffer (NuPage LDS sample buffer, Invitrogen NP0007) - in 4 °C below Biacore | ||
*Prestained protein ladder (NEB P7711S) | *MOPS buffer (Invitrogen NP0001) - above gel bench | ||
*Protein loading buffer | *Nitrocellulose membrane - in drawer below gel bench | ||
*MOPS buffer (Invitrogen NP0001) | *Blotting pads - in drawer below gel bench | ||
*Nitrocellulose membrane | *Transfer buffer (Invitrogen NP0006-1) - above gel bench | ||
*Blotting pads | |||
*Transfer buffer (Invitrogen NP0006-1) | |||
*Methanol | *Methanol | ||
*Anti-V5-HRP antibody (Invitrogen R961-25) | *Anti-V5-HRP antibody (Invitrogen R961-25) - if HA-tagged, anti-HA-HRP antibody (Abcam ab1188) - aliquoted in ME bay freezer | ||
*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 - 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 | |||
==Procedure== | ==Procedure== | ||
===Lysis=== | ===Lysis=== | ||
#Grow culture (5mL works well) in appropriate (generally dropout) media | #Grow culture (5mL works well) in appropriate (generally dropout) media | ||
#Pre-weigh an appropriate number of eppendorf tubes | #Pre-weigh an appropriate number of eppendorf tubes | ||
#*The variation in weight of the eppendorf tubes is significantly larger than the weight of the pellet | #*The variation in weight of the eppendorf tubes is significantly larger than the weight of the pellet | ||
# | #Pellet cells at 3000g, 4 °C for 5 minutes | ||
#Repellet | #Reweigh tubes, resuspend at 100 uL/mg in 0.1 M NaOH | ||
# | #Incubate at RT for 5 min | ||
# | #Repellet cells, resuspend at 25 uL/mg in 1x Laemmli buffer and transfer 50uL to a PCR tube | ||
#Heat at 95 °C for 3 min | |||
#Repellet, max speed, RT, 5 min | |||
#Remove supernatant | |||
#*Samples can now be stored at RT overnight | |||
===SDS-PAGE=== | ===SDS-PAGE=== | ||
# | #Take 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid) | ||
#Prep pre-cast SDS-PAGE gel | |||
# | |||
#Load 20uL of each sample | #Load 20uL of each sample | ||
#Run gel with MOPS running buffer, 150V, ~1hr | #Run gel with MOPS running buffer, 150V, ~1hr | ||
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==References== | ==References== | ||
Kushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860. | |||
==Contact== | ==Contact== |
Revision as of 14:10, 29 April 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 |