Hi all,

My lab is new to Western Blotting.

We are using NuPage System for Gel electrophoresis and Western Blotting as follows

Electrophoresis with the XCell SureLock™ Mini-Cell.

· NuPAGE® Novex® Bis-Tris Gel (SDS-PAGE) 10%, 1mm

· NuPAGE® LDS Sample Buffer

· NuPAGE® Reducing Agent

· NuPAGE® Antioxidant

· 1 x MOPS-SDS Running Buffer (Boston BioProducts, BP-178)

· Biorad PowerPac Universal with Novex™ Power Supply Adapters

· Single gel

· 200 Volts constant for 50 minutes

For Electrophoresis, the current starts at 100-115 mA and ends at 60-70 mA as expected according to the NuPAGE® Technical Guide. Gels look fantastic when stained by Coomassie Blue-R-250. Yay!

However, when we try to transfer protein to a PVDF membrane (no staining)....

Western Transfer Using the XCell II™ Blot Module with the XCell SureLock™ Mini-Cell,

· G-Biosciences 786018PV PVDF MEMBRANE (7.5 X 8.5CM), PORE SIZE 0.2µM (activated in methanol 30 seconds to 2 minutes)

· XCell II™ Blot Module Blotting Pads

· Whatman GB004 blotting paper

· NuPAGE® Antioxidant

· 1 x Bis Tris Transfer Buffer (Boston BioProducts, BP-193)

· Biorad PowerPac Universal with Novex™ Power Supply Adapters

· Single gel transferred

· 30 Volts constant for 1 hour (mini cell set in ice bath)

The current starts at 170 mA as expected, but then it increases slowly to >400 mA at the 1 hour time point. According to the NuPAGE® Technical Guide, the current should decrease from 170 mA at the start to 110 mA during the run.

Any ideas why the observed END current is not matching the predictions from the technical guide?

(Page 38NuPAGE® Technical Guide General information and protocols for using the

NuPAGE® electrophoresis system Rev. date: 29 October 2010 Manual part no. IM-1001)