Blotting for large V5-tagged proteins in S. cerevisiae
Y-PER (Pierce)
Halt EDTA-free Protease Inhibitor (Pierce)
NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX)
Prestained protein ladder (NEB P7711S)
Protein loading buffer
MOPS buffer (Invitrogen NP0001)
Nitrocellulose membrane
Blotting pads
Transfer buffer (Invitrogen NP0006-1)
Methanol
Anti-V5-HRP antibody (Invitrogen R961-25)
Chemiluminescence detection kit (Pierce)
Grow culture (5mL works well) in appropriate (generally dropout) media
Pellet cells at 3000g, 4°C for 5 minutes
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
Pour off the supernatant, resuspend in 1mL water. Transfer to a pre-weighed 1.5mL tube
Repellet as before, reweigh, and resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor)
Agitate at RT for 20min
Pellet at 14000g for 10 minutes
Remove 20uL of lysate supernatant, mix with 5uL of 5x loading buffer
Also take 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid)
Boil samples while prepping precast SDS-PAGE gel
Load 20uL of each sample
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
Cut out membrane slightly larger than gel
Be careful not to touch membrane - use forceps.
Make 2x transfer buffer + 10% MeOH
Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes
Soak pads + membrane in 2x transfer buffer + 10% MeOH
Layer pad, membrane, gel, pad
Roll a pipet over stack to press out bubbles
Run 15V, 20 minutes
Check for transfer - did the (prestained) ladder transfer over?
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)
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)
Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking)
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.
SDS-Page: At this step I usually take 40uL of supernatant (and mix it with 10uL 5x loading buffer), then remove the rest and resuspend the pellet in 200uL 60% glycerol. Then I take 40uL of the resuspended pellet and mix it with 10uL 5x loading buffer and then continue following the protocol. This pellet resuspention isn''t optimized; the pellet fraction usually ends up looking like a dot/blob when the western is imaged - but you can at least get an idea of where your protein is in the cell (supernatant = soluble, pellet = probably membrane bound). -Isis
For membrane-associated proteins, I sometimes resuspend the pellet in 2% SDS, boil it, spin it down again, and load the supernatant. That can pull out weakly associated proteins.
Semi-Dry Transfer: I just use 1x transfer buffer (with no methanol) and don''t pre-equilibrate the gel. I think I can get away with this because my proteins are relatively small (at least compared to Josh''s). Also, for some reason the voltage doesn''t always stay at 15V after I set it, so I always go back and double check and adjust after a few minutes of running. -Isis
On the high voltage source, you set upper limits on both the current and voltage. For the semidry transfer you have (initially) a very low voltage and very high current. Make sure the current limit is wide open, and the voltage limit is what''s keeping the voltage at 15V. If the current limit is initially responsible for keeping the system at 15V then the voltage will drift up over time. -Josh