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

Procedure

Sample preparation

Wells can accommodate 25μL loading volume.

  1. Each sample should contain
    • 13 μL protein sample (max 0.5μg per band)
    • 5 μL NuPAGE LDS Sample Buffer
    • 2 μL NuPAGE Reducing Agent
  2. Heat samples at 70°C for 10 mins.

Running buffer preparation

  1. Prepare 1000mL 1X NuPAGE SDS running buffer
    • 50mL 20X MES Running Buffer
    • 950mL deionized water
  2. Mix well.
  3. Set aside 200mL buffer for use in Upper (Inner) buffer chamber).
    • Add 500μL NuPAGE Antioxidant immediately before use.
    • Mix well.

References

  1. NuPAGE Technical Guide [NuPAGETechnicalGuide]
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