Smolke:Protocols/Western: Difference between revisions
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==Overview== | ==Overview== | ||
Blotting for V5-tagged proteins in ''S. cerevisiae'' | Blotting for large V5-tagged proteins in ''S. cerevisiae'' | ||
==Materials== | ==Materials== | ||
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==Procedure== | ==Procedure== | ||
===Lysis=== | ===Lysis=== | ||
#Grow culture (between 5 and 25mL works well) | #Grow culture (between 5 and 25mL works well) in appropriate (generally dropout) media | ||
#Pellet cells at 3000g, 4°C for 5 minutes | #Pellet cells at 3000g, 4°C for 5 minutes | ||
#Pour off supernatant, weigh, resuspend in water at 1 mg/uL | #Pour off supernatant, weigh, resuspend in water at 1 mg/uL | ||
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#Load 25uL of each sample | #Load 25uL of each sample | ||
#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 | |||
#Crack open gel, trim off top (wells) and bottom of gel with razor | |||
#*Can trim gel down further if all lanes weren't used | |||
===Semi-dry Transfer=== | ===Semi-dry Transfer=== | ||
#Cut out membrane slightly larger than gel | |||
#*Be careful not to touch membrane - use forceps. | |||
#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 | ||
#Soak pads + membrane in 2x transfer buffer + 10% MeOH | #Soak pads + membrane in 2x transfer buffer + 10% MeOH | ||
#Layer pad, membrane, gel, pad | #Layer pad, membrane, gel, pad | ||
#Roll a pipet over stack to press out bubbles | |||
#Run 15V, 20 minutes | #Run 15V, 20 minutes | ||
#*Check for transfer - did the (prestained) ladder transfer over? | |||
===Blotting=== | ===Blotting=== | ||
#Incubate membrane with 1x TBST + 5% milk, >1hr (rocking) | #Incubate membrane with 1x TBST + 5% milk, >1hr (rocking) | ||
#*Make sure there are no clumps in the milk (particularly a problem if you store the TBST+milk) | |||
#Wash twice with 1x TBST, 5 minutes (rocking) | #Wash twice with 1x TBST, 5 minutes (rocking) | ||
#Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking) | #Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking) | ||
#*Can go overnight at 4°C for higher sensitivity | |||
#Wash twice with 1x TBST, 5 minutes (rocking) | #Wash twice with 1x TBST, 5 minutes (rocking) | ||
#Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking) | #Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking) | ||
#Image | #Take membrane out, place on plastic wrap, blot lightly with Kimwipe. Then fold over plastic wrap to cover (squeeze out bubbles) | ||
#Image membrane | |||
#*Use gel imager on chemiluminescence setting. Set for 5-100 s, in 5s increments | |||
#*It's also useful to take another picture in white light (without moving membrane) to image the ladder. | |||
==Notes== | ==Notes== |
Revision as of 14:44, 25 January 2009
Still a work in progress OverviewBlotting for large V5-tagged proteins in S. cerevisiae Materials
ProcedureLysis
SDS-PAGE
Semi-dry Transfer
Blotting
NotesReferencesContact |