Smolke:Protocols/Western
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{{Smolke_Top}} | {{Smolke_Top}} | ||
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| + | Still a work in progress | ||
==Overview== | ==Overview== | ||
| - | + | Blotting for V5-tagged proteins in ''S. cerevisiae'' | |
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==Materials== | ==Materials== | ||
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==Procedure== | ==Procedure== | ||
| + | ===Lysis=== | ||
| + | #Grow 25mL culture to saturation in appropriate (generally dropout) media. | ||
| + | #Pellet cells at 3000g, 4°C for 5 minutes | ||
| + | #Pour off supernatant, weigh, resuspend in water at 1 mg/mL. | ||
| + | #Transfer 100uL to a 1.5mL tube, repellet as before | ||
| + | #Reweigh, resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor) | ||
| + | #Agitate at RT for 20min | ||
| + | #Pellet at 14000g, 4°C for 10 minutes | ||
| + | |||
| + | ===SDS-PAGE=== | ||
| + | #Remove 40uL of lysate supernatant, mix with 10uL of 5x loading buffer | ||
| + | #*Also take 5uL prestained ladder (thaw and vortex first) | ||
| + | #Boil samples while prepping precast SDS-PAGE gel | ||
| + | #Load 25uL of each sample | ||
| + | #Run gel with MOPS running buffer, 150V, ~1hr | ||
| + | |||
| + | ===Transfer=== | ||
| + | #Make 2x transfer buffer + 10% MeOH | ||
| + | #Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes | ||
| + | #Soak pags + membrane in 2x transfer buffer + 10% MeOH | ||
| + | #Layer pad, membrane, gel, pad | ||
| + | #Run 15V, 20 minutes | ||
| + | ===Blotting=== | ||
| + | #Incubate membrane with 1x TBST + 5% milk, >1hr (rocking) | ||
| + | #Wash twice with 1x TBST, 5 minutes (rocking) | ||
| + | #Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking) | ||
| + | #Wash twice with 1x TBST, 5 minutes (rocking) | ||
| + | #Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking) | ||
| + | #Image | ||
==Notes== | ==Notes== | ||
Revision as of 17:26, 23 January 2009
