Smolke:Protocols/Western

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Still a work in progress
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==Overview==
==Overview==
-
Blotting for V5-tagged proteins in ''S. cerevisiae''
+
Blotting for large V5-tagged proteins in ''S. cerevisiae''
==Materials==
==Materials==
-
*Y-PER ([http://www.piercenet.com/products/browse.cfm?fldID=06010430 Pierce])
+
*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) - in cold room
-
*Halt EDTA-free Protease Inhibitor ([http://www.piercenet.com/Products/Browse.cfm?fldID=02040806 Pierce])
+
*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 (Invitrogen LC5800)
+
*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)
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*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 25mL culture to saturation in appropriate (generally dropout) media.
+
#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
-
#Pour off supernatant, weigh, resuspend in water at 1 mg/uL.
+
#*The variation in weight of the eppendorf tubes is significantly larger than the weight of the pellet
-
#Transfer 100uL to a 1.5mL tube, repellet as before
+
#Pellet cells at 3000g, 4 °C for 5 minutes
-
#Reweigh, resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor)
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#Reweigh tubes, resuspend at 100 uL/mg in 0.1 M NaOH
-
#Agitate at RT for 20min
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#Incubate at RT for 5 min
-
#Pellet at 14000g, 4°C for 10 minutes
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#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===
-
#Remove 40uL of lysate supernatant, mix with 10uL of 5x loading buffer
+
#Take 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid)
-
#*Also take 5uL prestained ladder (thaw and vortex first)
+
#Prep pre-cast SDS-PAGE gel
-
#Boil samples while prepping precast SDS-PAGE gel
+
#Load 20uL of each sample
-
#Load 25uL of each sample
+
#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
 +
#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===
===Semi-dry Transfer===
 +
#Cut out membrane slightly larger than gel
 +
#*Be careful not to touch membrane - use forceps.
#Make 2x transfer buffer + 10% MeOH
#Make 2x transfer buffer + 10% MeOH
#Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes
#Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes
#Soak pads + membrane in 2x transfer buffer + 10% MeOH
#Soak pads + membrane in 2x transfer buffer + 10% MeOH
#Layer pad, membrane, gel, pad
#Layer pad, membrane, gel, pad
 +
#Roll a pipet over stack to press out bubbles
#Run 15V, 20 minutes
#Run 15V, 20 minutes
 +
#*Check for transfer - did the (prestained) ladder transfer over?
===Blotting===
===Blotting===
#Incubate membrane with 1x TBST + 5% milk, >1hr (rocking)
#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)
#Wash twice with 1x TBST, 5 minutes (rocking)
#Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (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)
#Wash twice with 1x TBST, 5 minutes (rocking)
#Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking)
#Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking)
-
#Image
+
#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==
==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==

Current revision

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Contents

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) - aliquoted in ME bay freezer
  • Protein loading buffer (NuPage LDS sample buffer, Invitrogen NP0007) - in 4 °C below Biacore
  • MOPS buffer (Invitrogen NP0001) - above gel bench
  • 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 ME bay freezer
  • Chemiluminescence detection kit (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

Lysis

  1. Grow culture (5mL works well) in appropriate (generally dropout) media
  2. 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
  3. Pellet cells at 3000g, 4 °C for 5 minutes
  4. Reweigh tubes, resuspend at 100 uL/mg in 0.1 M NaOH
  5. Incubate at RT for 5 min
  6. Repellet cells, resuspend at 25 uL/mg in 1x Laemmli buffer and transfer 50uL to a PCR tube
  7. Heat at 95 °C for 3 min
  8. Repellet, max speed, RT, 5 min
  9. Remove supernatant
    • 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
    • Run until dye front is near bottom of gel
  5. 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

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

Blotting

  1. 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)
  2. Wash twice with 1x TBST, 5 minutes (rocking)
  3. Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking)
    • Can go overnight at 4°C for higher sensitivity
  4. Wash twice with 1x TBST, 5 minutes (rocking)
  5. Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking)
  6. Take membrane out, place on plastic wrap, blot lightly with Kimwipe. Then fold over plastic wrap to cover (squeeze out bubbles)
  7. 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

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