Smolke:Protocols/Western

Overview
Blotting for large V5-tagged proteins in S. cerevisiae

Materials

 * 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)

Lysis

 * 1) Grow culture (5mL works well) in appropriate (generally dropout) media
 * 2) Pre-weigh an appropriate number of eppendorf tubes
 * 3) *The variation in weight of the eppendorf tubes is significantly larger than the weight of the pellet
 * 4) Pellet cells at 3000g, 4 °C for 5 minutes
 * 5) Reweigh tubes, resuspend at 100 uL/mg in 0.1 M NaOH
 * 6) Incubate at RT for 5 min
 * 7) Repellet cells, resuspend at 25 uL/mg in 1x Laemmli buffer and transfer 50uL to a PCR tube
 * 8) Heat at 95 °C for 3 min
 * 9) Repellet, max speed, RT, 5 min
 * 10) Remove supernatant
 * 11) *Samples can now be stored at RT overnight

SDS-PAGE

 * 1) Take 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid)
 * 2) Prep pre-cast SDS-PAGE gel
 * 3) Load 20uL of each sample
 * 4) Run gel with MOPS running buffer, 150V, ~1hr
 * 5) *Run until dye front is near bottom of gel
 * 6) Crack open gel, trim off top (wells) and bottom of gel with razor
 * 7) *Can trim gel down further if all lanes weren't used

Semi-dry Transfer

 * 1) Cut out membrane slightly larger than gel
 * 2) *Be careful not to touch membrane - use forceps.
 * 3) Make 2x transfer buffer + 10% MeOH
 * 4) Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes
 * 5) Soak pads + membrane in 2x transfer buffer + 10% MeOH
 * 6) Layer pad, membrane, gel, pad
 * 7) Roll a pipet over stack to press out bubbles
 * 8) Run 15V, 20 minutes
 * 9) *Check for transfer - did the (prestained) ladder transfer over?

Blotting

 * 1) Incubate membrane with 1x TBST + 5% milk, >1hr (rocking)
 * 2) *Make sure there are no clumps in the milk (particularly a problem if you store the TBST+milk)
 * 3) Wash twice with 1x TBST, 5 minutes (rocking)
 * 4) Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking)
 * 5) *Can go overnight at 4°C for higher sensitivity
 * 6) Wash twice with 1x TBST, 5 minutes (rocking)
 * 7) Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking)
 * 8) Take membrane out, place on plastic wrap, blot lightly with Kimwipe. Then fold over plastic wrap to cover (squeeze out bubbles)
 * 9) Image membrane
 * 10) *Use gel imager on chemiluminescence setting. Set for 5-100 s, in 5s increments
 * 11) *It's also useful to take another picture in white light (without moving membrane) to image the ladder.

Contact
Josh Michener