Smolke:Protocols/Western

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{{Smolke_Top}}
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==Overview==
==Overview==
-
 
+
Blotting for large V5-tagged proteins in ''S. cerevisiae''
-
Replace this sentence with a brief description of the protocol and its goal.
+
==Materials==
==Materials==
-
List reagents, supplies and equipment necessary to perform the protocol here.  For those materials which have their own OWW pages, link to that page.  Alternatively, links to the suppliers' page on that material are also appropriate.
+
*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) - in cold room
-
 
+
*Prestained protein ladder (NEB P7711S) - aliquoted in ME bay freezer
-
*supply 1 (i.e. tubes of a certain size? spreaders?)
+
*Protein loading buffer (NuPage LDS sample buffer, Invitrogen NP0007) - in 4 °C below Biacore
-
*reagent 1
+
*MOPS buffer (Invitrogen NP0001) - above gel bench
-
*X μL reagent 2
+
*Nitrocellulose membrane - in drawer below gel bench
-
**component A (reagent 2 is made up of multiple components)
+
*Blotting pads - in drawer below gel bench
-
**component B
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*Transfer buffer (Invitrogen NP0006-1) - above gel bench
-
*equipment 1
+
*Methanol
-
*equipment 2
+
*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]) - in Steph's drawer
 +
*BSA fraction V or dried milk for blocking - in cold room
 +
*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) - in cold room
==Procedure==
==Procedure==
-
#Step 1
+
===Lysis===
-
#Step 2
+
#Grow culture (5mL works well) in appropriate (generally dropout) media
-
#*Step 2 has some additional information that goes with it.  i.e. Keep at 4°C.
+
#Pre-weigh an appropriate number of eppendorf tubes
-
#Step 3
+
#*The variation in weight of the eppendorf tubes is significantly larger than the weight of the pellet
-
##Step 3 has multiple sub-steps within it.
+
#Pellet cells at 3000g, 4 °C for 5 minutes
-
##Enumerate each of those.
+
#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===
 +
#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
 +
#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
 +
#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==
==Notes==
-
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
+
*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
-
#List troubleshooting tips here.
+
**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.
-
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
+
-
#Anecdotal observations that might be of use to others can also be posted here.
+
-
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
+
*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==
-
'''Relevant papers and books'''
+
Kushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860.
-
<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
+
-
<biblio>
+
-
#Goldbeter-PNAS-1981 pmid=6947258
+
-
#Jacob-JMB-1961 pmid=13718526
+
-
#Ptashne-Genetic-Switch isbn=0879697164
+
-
</biblio>
+
==Contact==
==Contact==
-
 
+
[[Josh Michener]]
[[Category:Protocol]]
[[Category:Protocol]]
[[Category:In vitro]]
[[Category:In vitro]]
[[Category:Yeast]]
[[Category:Yeast]]
-
-->
 

Revision as of 20:48, 29 January 2014

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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 Steph's drawer
  • BSA fraction V or dried milk for blocking - in cold room
  • 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) - 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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