Inconsistent Western blotting results - (Jun/24/2008 )
I would like to know what could be the steps in western blot that went wrong as my first results and second results were inconsistent?
My first results showed downregulation. However, when I confirm it again, it remained the same. (no downregulation nor upregulation).
Thanks in advance.
was this a re-run of exactly the same protein extracts, with all the same reagents and supplies? if so, it's possible that your extracts have experienced some degradation? but there could be many reasons.
please provide more detail - and it's quite helpful if you can post a pic of the blot
Ya. It is the same protein from the same badge of cells.
The pic shows my first attempt results. My second attempt results shows equal band for all samples.
For my first attempt, I had placed that membrane with the other membrane rolled in a tube. But I can confirm there is no overlapping of the two membranes as when I was about to take them out, they were separately attached to the tube.
Whereas for my second attempt, I had only place one membrane rolled in a tube.
Therefore, I was wondering is it the depletion of the primary antibodies for my first attempt?
All other conditions were the same.
My actin was equal for both.
do you mean that you performed the second experiment in the same reagents, without making up any fresh batches of antibodies? if this is the case, I would think that yes, at least one of your reagents degraded or something between one blot and the next.
secondly - sample degradation could be a factor, especially if the relative detection in the second was much less
third - it's possible there was a problem with the gel transfer on the first one, giving uneven bands.