mayeck

Hi

Couple of days ago I tried my first Western Blotting and the result is pretty ugly. I proceeded with blotting as follows:
- Blocking over the weekend in 5% milk in TBST (with 0,02% sodium azide), 4 degrees with rocking
- 3 hour incubation with mouse monoclonal anti-MPM2 1:1000 (same buffer)
- 1 hour secondary Cell Signalling 1:1000
and developed film with Pierce ECL
Result is shown in picture 1, background is very high and bands from MPM-2 are visible only in one lane.

My reasoning was that 3 days blocking time with milk and tween wasn't very bright idea since MPM-2 detects phophoepitope (and also I've heard, that 4 degrees incubation should be performed without tween). So I dehybridised membrane with standard 0,2 M NaOH and tried to turn whole procedure around, this time:
- Blocking 1 hour at room temperature (milk in TBST, of course no azide here)
- 1 hour goat monoclonal antibody picted somewhat at random from the shelf, established to work earlier by a friend, 1:1000 (Santa Cruz)
- 1 hour Jackson antigoat, 1:10.000
Result (picture 2) seems even worse and the whole blot is very dirty. However the most surprising part is that in this blot bands (although very faint, probably too short primary antibody exposure) can be seen in all 6 lanes.

I'm completely lost at what have happened here and any suggestion should be helpful. Some silly basic mistakes not impossible.

 western 01.jpg   67.51K   54 downloads  western 02.jpg   76.89K   57 downloads