Knight:NuPAGE electrophoresis: Difference between revisions
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==Purpose== | ==Purpose== | ||
To run a protein gel. Note that a preferable version of this protocol is available at [[Sauer:bis-Tris SDS-PAGE, the very best]] (recommended by Kathleen). But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system. | To run a denaturing protein gel. Note that a preferable version of this protocol is available at [[Sauer:bis-Tris SDS-PAGE, the very best]] (recommended by Kathleen). But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system. | ||
==Materials== | ==Materials== | ||
*NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well | *NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well | ||
*[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=792& XCell ''SureLock'' Mini-Cell electrophoresis apparatus] | *[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=792& XCell ''SureLock'' Mini-Cell electrophoresis apparatus] | ||
*20X MES Running Buffer | |||
*NuPAGE LDS Sample Buffer (4X) | |||
*NuPAGE Reducing Agent (10X) | |||
*NuPAGE Antioxidant | |||
==Procedure== | ==Procedure== | ||
===Sample preparation=== | |||
Wells can accommodate 25μL loading volume. | |||
#Each sample should contain | |||
#*13 μL protein sample (max 0.5μg per band) | |||
#*5 μL NuPAGE LDS Sample Buffer | |||
#*2 μL NuPAGE Reducing Agent | |||
#Heat samples at 70°C for 10 mins. | |||
===Running buffer preparation=== | |||
#Prepare 1000mL 1X NuPAGE SDS running buffer | |||
#*50mL 20X MES Running Buffer | |||
#*950mL deionized water | |||
#Mix well. | |||
#Set aside 200mL buffer for use in Upper (Inner) buffer chamber). | |||
#*Add 500μL NuPAGE Antioxidant immediately before use. | |||
#*Mix well. | |||
==References== | ==References== | ||
Revision as of 10:56, 17 July 2006
In progress! For a better version of this protocol, see Sauer:bis-Tris SDS-PAGE, the very best.
Purpose
To run a denaturing protein gel. Note that a preferable version of this protocol is available at Sauer:bis-Tris SDS-PAGE, the very best (recommended by Kathleen). But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system.
Materials
- NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well
- XCell SureLock Mini-Cell electrophoresis apparatus
- 20X MES Running Buffer
- NuPAGE LDS Sample Buffer (4X)
- NuPAGE Reducing Agent (10X)
- NuPAGE Antioxidant
Procedure
Sample preparation
Wells can accommodate 25μL loading volume.
- Each sample should contain
- 13 μL protein sample (max 0.5μg per band)
- 5 μL NuPAGE LDS Sample Buffer
- 2 μL NuPAGE Reducing Agent
- Heat samples at 70°C for 10 mins.
Running buffer preparation
- Prepare 1000mL 1X NuPAGE SDS running buffer
- 50mL 20X MES Running Buffer
- 950mL deionized water
- Mix well.
- Set aside 200mL buffer for use in Upper (Inner) buffer chamber).
- Add 500μL NuPAGE Antioxidant immediately before use.
- Mix well.
