densitometry on western blots

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum At hotmail.com)
Wed Dec 13 10:11:26 EST 2006


DK wrote:

> Perhaps you encountered some bad  M-L implementations? 

Gnuplot and Sigma-Plot. No idea how good the latter is, but the former
is open source and has probably been honed close to optimal over the
last 15 years or so.

> No arguing here. But you have to admit that five orders of signal, 
> measured with 1% error over the entire range is something rather
> astoundingly exceptional for biologists. 

Not if kinetics of multiple site enzymes is done properly. At least in
my own field, membrane transporters, both P-type and ABC-type pumps show
high affinity sites with Kd ~ 100 nM and low affinity ones with Kd ~ 300
uM, respectively. In F-type ATPases things are even more complicated.
Since one should measure from 1/10 Kd to 10 Kd, that means you have to
cover a range from 10 nM to 3 mM. And then you see some substrate
inhibition, so you go on to 10 mM or so.

This is not technically difficult if you work with a constant amount of
radioactivity in each assay, that is varying specific activity. You also
have to adjust the amount of enzyme in the assay to stay in the linear
range, but surprisingly little (about a factor of 2 or 3). Then you get
a constant relative error which depends on your pipetting skills. 1-2%
is quite achivable. Obviously, with constant specific activity (and
hence constant absolute standard deviation) such a range could not be
covered. 

Of course, non specialists in enzyme kinetics don't follow the rules. As
a consequence, several papers from well regarded laboratories claimed
that the ABC-transporter Mdr1 has only a single ATP-site. 




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