Smolke:Protocols/Western: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
(10 intermediate revisions by 3 users not shown) | |||
Line 6: | Line 6: | ||
==Materials== | ==Materials== | ||
*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) - in cold room | |||
*Prestained protein ladder (NEB P7711S) - aliquoted in ME bay freezer | |||
*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) | *Protein loading buffer (NuPage LDS sample buffer, Invitrogen NP0007) - in 4 °C below Biacore | ||
*Prestained protein ladder ( | *MOPS buffer (Invitrogen NP0001) - above gel bench | ||
*Protein loading buffer | *Nitrocellulose membrane - in drawer below gel bench | ||
*MOPS buffer (Invitrogen NP0001) | *Blotting pads - in drawer below gel bench | ||
*Nitrocellulose membrane | *Transfer buffer (Invitrogen NP0006-1) - above gel bench | ||
*Blotting pads | |||
*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) - aliquoted in ME bay freezer | ||
*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]) - in drawer below gel bench | ||
*BSA fraction V or dried milk for blocking - 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== | ==Procedure== | ||
===Lysis=== | ===Lysis=== | ||
#Grow culture ( | #Grow culture (5mL works well) in appropriate (generally dropout) media | ||
# | #Pre-weigh an appropriate number of eppendorf tubes | ||
#*The | #*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 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 | |||
#Run gel with MOPS running buffer, 150V, ~1hr | #Run gel with MOPS running buffer, 150V, ~1hr | ||
#*Run until dye front is near bottom of gel | #*Run until dye front is near bottom of gel | ||
Line 65: | Line 67: | ||
==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 | |||
**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== | ==References== | ||
Kushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860. | |||
==Contact== | ==Contact== |
Revision as of 14:10, 29 April 2014
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 |