Introduction

Instructions are provided below for electrophoresis of Novex® Tricine Gels using the XCell SureLock™ Mini-Cell. For detailed instructions, refer to the Novex® Pre-Cast Gel Electrophoresis Guide available at www.invitrogen.com or contact Technical Support.

Prepare Samples

Reagent Reduced Sample Non-reduced Sample
Sample x μl x μl
Novex® Tricine SDS Sample Buffer (2X) 5 μl 5 μl
NuPAGE® Reducing Agent (10X) 1 μl --
Deionized Water to 4 μl to 5 μl
Total Volume 10 μl 10 μl

Heat samples at 85°C for 2 minutes.

Prepare Running Buffer

Prepare 1X Tricine SDS Running Buffer by adding 100 ml of 10X Novex® Tricine SDS Running Buffer to 900 ml of deionized water.

Load Sample

Load the appropriate concentration and volume of your protein sample on the gel.

Load Buffer

Fill the Upper Buffer Chamber with 200 ml and the Lower Buffer Chamber with 600 ml of 1X Tricine SDS Running Buffer.

Run Conditions

Voltage:   125 V constant
Run Time:   90 minutes (dependent on gel percentage)
Expected Current:  80 mA/gel (start); 40 mA/gel (end)

Blotting Conditions

For blotting Tricine gels, use 1X Tris-Glycine Transfer Buffer with 20% methanol. Perform transfer with nitrocellulose or PVDF membranes at 25 V constant for 1–2 hours using the XCell II™ Blot Module. The expected start current is 100 mA.

Alternate Transfer Buffers

The Tris-Glycine Transfer Buffer interferes with protein sequencing. If you are performing protein sequencing, use 1X NuPAGE® Transfer Buffer or 0.5X TBE Transfer Buffer for blotting. The NuPAGE® Transfer Buffer protects against modification of the amino acid side chains and is compatible with N-terminal protein sequencing using Edman degradation.
TOP
 IM-6002  Version C   1-Oct-2008