mRNA level and protein level correlation?
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
>(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!).
>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.
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