Oscillation on my Western Blot results - (Jun/08/2011 )

Hi,

I would like to get some advice about Western Blotting technique. My research is concerned about A₁ adenosine receptor. The results of my westerns are never stable…I’ve been observing an oscillation in the signal obtained! I got these three situations: strong signal, weak signal and no signal.

My protocol is as follows:

-Homogeneization of samples in 50mM Tris-HCl pH 7.4 with protein inhibitors.
-Samples were diluted into two volumes of a urea lysis buffer and incubated for 2 hours at 37ºC.
-10% SDS-PAGE gel, 92V (stacking) – 135V (running)
-Wet transfer, 350mA (2h30) for two gels
-Check transfer with Ponceau S stain (result: ok!)
-Block in 5% non-fat dry milk 1x PBS-TW (2h)
-Primary antibody incubation overnight in 3% non-fat dry milk 1x PBS-TW followed by 3x15’ washes with 1x PBS-TW
-Secondary antibody incubation for 2 hours at room temperature with agitation followed by 3x15’ washes with 1x PBS-TW
-The membranes were developed with chemiluminescent reagent ECL Plus

Any help would be appreciated,
Thanks in advance!

Fresh dilutions of the antibody each time?

fresh protease inhibitors each time?

bob1 on Thu Jun 9 09:21:50 2011 said:

Yes, since I've noticed oscillations on results, I prepare everything fresh and I'm still observing these variations...

Perhaps it is something to do with the sample prep rather than the western as your protocol looks fine. Perhaps you are keeping some samples on ice for longer than others, or doing more freeze/thaw cycles?

As a note: You will sometimes see a difference in signal strength between two identical gels when blotting both in the same tank. I think this is due to some interference from one of the blots, as it always seems to be the blot closest to the black terminal that has stronger signal...