Smolke:Protocols/Western: Difference between revisions
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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 |