Knight:NuPAGE electrophoresis
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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 13:56, 17 July 2006
In progress! For a better version of this protocol, see Sauer:bis-Tris SDS-PAGE, the very best.
Contents |
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.
