a question of sufficient n's

A.F. Simpson AFS7 at le.ac.uk
Mon Aug 16 20:05:12 EST 1999

John Hines wrote:

> In the experiment, 8 mice are treated with the drug for the prescribed
> time and dosage and 8 mice are treated with vehicle.  Their livers are
> pooled according to treatment group (drug or control) and homogenized.
> SDS-PAGE is done and the blots are performed.
> Now, if the scientist runs a 7 lane gel, where:
> lane 1 = MW markers
> lane 2, 4, 6 = control homogenate 
> lane 3, 5, 7 = drug treated homogenate
> and then blots the entire piece of nitrocellulose for the level of target
> enzyme, is that 3 replicate measurements (comparing lane 2 vs. 3; 4 vs. 5;
> 6 vs. 7) ??   Or is that an n of 3?
> or second scenario:
> if the scientist runs a 3 lane gel, where:
> lane 1 = MW markers
> lane 2 = control homogenate
> lane 3 = drug treated homogenate
> and blots for the enzyme.  And then runs a second identical gel next week and
> blots that; and then runs a third identical gel the following week and
> blots that; is that 3 replicate measurements (remember, the very same
> homogenates were used in all the blots), or is that an n of 3??

(sorry for the extensive quote, but I couldn't find a way of trimming it
and still have it make sense for people who hadn't seen the firth post.)

In both cases I would say n=1, because you only have one sample for each
condition - the homogenised liver pools for treament and control.  You
have no replication because you only performed the actual treatment
experiment once.

What the two proceedures do give you is an estimte of the repeatability
of the measurement ("3 replicate measurements").  The first protocol
control for variation of loading etc within a single western, the second
for the repeatability of the western.  I would say that triplicate
measurements are the minimum, especially for quantitating by Western
which is not especially accurate.

Personally, I would do at least three separate westerns, and I would
load a dilution series of treatment and control on each one to make sure
I wasn't overloading.

But at the end of the day, n=1.

> John
> hines at pharm.med.upenn.edu


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