Ponceau S and Coomasie do not correlate to b actin signal - (May/15/2010 )
Dukey on Jun 7 2010, 02:15 PM said:
I would suspect an extraction problem rather than a drastic loading issue. Whilst I agree with bob1 that there is differences in the load, in my opinion it is certainly not sufficient to explain the variability in the WB that you see. For example, in several lanes (i.e. lane 5) there is almost a complete loss of the actin but that is clearly not the case in the ponceau, even if you look at the 45 kDa region. You may need to look at your extraction/tissue collection procedure to troubleshoot this.
Thank you for your answer.
I have been asking around a lot lately and it seems that I am not the only one who has these issues. Maybe not to that extent but alla the fellows that I have asked and they are dealing with tissue samples seem to have this kind of problem.
Now, the odd thing is that I am handling all my samples (retinas) in the same manner and while I have huge discrepancied in the b actin signal, my protein of interest is not beeing affected that much.
Another issue is the fact that each lane is the sample from a retina from a different animal, so there is no way that the relative amount of b actin is the same between different animals. That would have been the case if I was supposed to take the contralateral eye and run a gel.
But even in that case I would have trouble between that different animal groups.
One thing I could do is to run a gel and see the differences and then adjust my loading according to the b actin signal i.e. load from each sample more or less according to the signal I get. I have already tried that in some older experiements and it gave me some results. Still I am not quite sure if this is the right way to go, since I will be also manipulating my samples according to the b actin.