mRNA level and protein level correlation?

Peter Frank peter_frankde at yahoo.de
Thu Mar 18 13:24:08 EST 2004


Michael L. Sullivan wrote:

>I think it is very hard to get quantitative data from a western both 
>because of the high degree of variation one gets within a single 
>blot, and also because western signals are very, very non-linear.

Very true. I also noticed that there is already a high degree of
variation between bands of a single blot.

>If you are making your measurements by densitometry of film rather than 
>more directly by using a lumi-imager, that throws yet another 
>variable into the mix.

I did both. Problem with the lumi-imager we have in our lab is that it
gives very faint bands compared to film - even after very long
exposure times.

>(I have a western blot right in front of me right now where a 3 fold 
>difference in loading resulted in only a 50%  increase in signal on 
>the western as measured on a lumi-imager under conditions where 
>the signal was not saturated!).

Very interesting.

>Minimally, I think one needs to run a dilution of each sample, and 
>probably run multiple blots to get believable numbers.

I haven't done dilutions (yet) but I have done multiple blots and
technical replicates on each blot. Then I evaluated the data
statistically. I am still not very confident in the absolute value of
the fold change but now I am at least confident enough about the
direction and the significance of the change.

After hearing about your experiences with quantitative Western blots I
am inclined to believe the answer actually lies in the lack of
quantitative accuracy rather than in biology.

Peter



More information about the Methods mailing list