Smolke:Protocols/Western: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
(4 intermediate revisions by 2 users not shown) | |||
Line 6: | Line 6: | ||
==Materials== | ==Materials== | ||
*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) | *NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) | ||
*Prestained protein ladder (NEB P7711S) | *Prestained protein ladder (NEB P7711S) | ||
*Protein loading buffer | *Protein loading buffer (NuPage LDS sample buffer, Invitrogen NP0007) | ||
*MOPS buffer (Invitrogen NP0001) | *MOPS buffer (Invitrogen NP0001) | ||
*Nitrocellulose membrane | *Nitrocellulose membrane | ||
Line 16: | Line 14: | ||
*Transfer buffer (Invitrogen NP0006-1) | *Transfer buffer (Invitrogen NP0006-1) | ||
*Methanol | *Methanol | ||
*Anti-V5-HRP antibody (Invitrogen R961-25) | *Anti-V5-HRP antibody (Invitrogen R961-25) - if HA-tagged, anti-HA-HRP antibody (Abcam ab1188) | ||
*Chemiluminescence detection kit ([http://www.piercenet.com/Products/Browse.cfm?fldID=01041101 Pierce]) | *Chemiluminescence detection kit ([http://www.piercenet.com/Products/Browse.cfm?fldID=01041101 Pierce]) | ||
*BSA fraction V or dried milk for blocking | |||
*10x TBST solution (80 g NaCl, 30 g Tris, 5 mL Tween 20, add ~850 mL MP water, adjust pH to 8 with concentrated HCl, fill to 1 L) | |||
==Procedure== | ==Procedure== | ||
===Lysis=== | ===Lysis=== | ||
#Grow culture ( | #Grow culture (5mL works well) in appropriate (generally dropout) media | ||
#Pellet cells at 3000g, | #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 | ||
#Repellet | #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 and transfer 50uL to a PCR tube | |||
#Heat at 95 °C for 3 min | |||
#Repellet, max speed, RT, 5 min | |||
#Remove supernatant | |||
#*Samples can now be stored at RT overnight | |||
===SDS-PAGE=== | ===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 | #Load 20uL of each sample | ||
#Run gel with MOPS running buffer, 150V, ~1hr | #Run gel with MOPS running buffer, 150V, ~1hr | ||
Line 64: | Line 68: | ||
==Notes== | ==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 | *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 | *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 | ||
Line 69: | Line 74: | ||
==References== | ==References== | ||
Kushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860. | |||
==Contact== | ==Contact== |
Revision as of 13:33, 5 July 2013
OverviewBlotting for large V5-tagged proteins in S. cerevisiae Materials
ProcedureLysis
SDS-PAGE
Semi-dry Transfer
Blotting
Notes
ReferencesKushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860. Contact |