Smolke:Protocols/Western

Home        Contact        Internal        Protocols        Lab Members        Publications        Research       

Overview Blotting for large V5-tagged proteins in S. cerevisiae Materials NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) - in cold room Prestained protein ladder (NEB P7711S) - 8 uL aliquots in freezer by LC-QQQ Protein loading buffer (NuPage LDS sample buffer, Invitrogen NP0007) - small bottle on shelf above gel bench, large bottle in deli fridge (let warm to room temp for LDS to redissolve, then aliquot into small bottle) MOPS running buffer (Invitrogen NP0001) - above gel bench (use MES instead for small proteins) Nitrocellulose membrane - in drawer below gel bench Blotting pads - in drawer below gel bench Transfer buffer (Invitrogen NP0006-1) - above gel bench Methanol Anti-V5-HRP antibody (Invitrogen R961-25) - if HA-tagged, anti-HA-HRP antibody (Abcam ab1188) - aliquoted in freezer by LC-QQQ Chemiluminescence detection kit (Pierce) - in drawer below gel bench BSA fraction V or dried milk for blocking - made from BSA in deli fridge, stock stored 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 Lysis Grow culture (5mL works well) in appropriate (generally dropout) media 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 Pellet cells at 3000g, 4 °C for 5 minutes 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 (or use LDS buffer instead) and transfer 50uL to a PCR tube Heat at 95 °C for 3 min (70 °C for 10 m for membrane-bound proteins) Repellet, max speed, RT, 5 min Remove supernatant Samples can now be stored at RT overnight 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 (20-40 ug total protein) 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 Cut out membrane slightly larger than gel Be careful not to touch membrane - use forceps. 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 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? Blotting 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. Notes 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 References Kushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860. Contact Josh Michener