problem with WB quantification

[Ossan]

Hi,

I'm having trouble in comparing the levels of a HA-tagged plant protein (130 kDa) in individual transgenic lines. The problem is that the even the calculated levels of a single protein sample give different values when loaded on different lanes of the gel. It looks like the efficiency of transfer varies from lane to lane in a very unreproducible manner (not a left to right gradient as sometimes observed).

I'm using a BioRad Tetra cell for gel run and blotting. I use Towbin buffer without MeOH but with 50 µM SDS and transfer to PVDF (Millipore) at 100V for 1h. I detect the protein with a-HA 3F10 from Roche at a 1:2000 dilution (recommended starting dil: 1:5000) and a 2nd Ab from Santa Cruz using the ECL Pierce Femto-enhancer substrate (otherwise no signal for the HA-tagged protein). Levels of the HA-tagged protein are normalized against CSN4 which runs at 45 kDa. Transfer of the 45 kDa region looks good in Ponceau staining and the calculated CSN4 values are pretty constant. Hence it looks like I'm having a problem with either the primary HA Ab or the transfer in the 130 kDa region?

I'd be grateful for any comments or suggestions on this problem!

thanks,
Frank