TNF alpha and IL 1b Western Blot - (Sep/15/2012 )

Hello im trying to do western blot for TNF alpha and for interleukin 1 beta in rat gastric tissue, both proteins are 17Kda proteins, I load 25-30 micrograms of my samples (1:1 with Laemmli) in a 15% Acrylamide 40% /Bisacrylamide Gel, the condition of my electrophoresis are 120 V for 90 minutes, for my transfer the condition are 310 mA for 90 minutes < i use the Biorad system for electrophoresis and transfer>, i block with 15% Milk /PBS tween 0.05% for 1 hr and put the Antibodies at dilution 1 : 200 in a 5% Milk/ PBS tween 0.05% buffer for overnigth, then 3 washes for 10 minutes with PBS- tween 0.05% and my secondary in a 1:3000 dilution in PBS tween 0.05% for 1 hr then 3 washes for 10 minutes with PBS- tween 0.05% (but the last only with pbs) and I reveal.

In the most of the cases I obtain non- specific union or I obtain two strong bands one at the top (250-100 kDa) and another of my molecular marker (aproximadly at 50-25 kDa). I would like some suggestions to remove my nonspecific union (i try milk at diferents conditions and albumin) or maybe it´s my sample. Thansk a lot

What I understood is that all you see are non specific bands and that you are not picking up your proteins of interest. You might want to see whether your proteins of interest have isoforms or whether they can form dimers. The other thing might be the primary antibody you are using. Try using one from a different company and see how the blots look.