In order for this experiment a complete repeat, the Western would have to be
repeated , as well; especially, since protein quantification by Western is
of questionable reliability.
What he is doing is to measure a triplicate of the same pooled sample, n=1.
As long as the livers are pooled into one homogenate, it will always be only
one sample pooled from 8 mice; the two options described do not change this.
The difference is only in the detection method. The first option allows to
monitor the variability of the same sample in one Western blot. The 2nd
option is slightly better, since the variability of the Western itself can
be monitored, as well. It allows to obtain more information on the accuracy
and precision of the detection method.
Achim
John Hines <hines at pharm.med.upenn.edu> wrote in message
news:hines-1208991713350001 at ovh173.vetschool.upenn.edu...
>> A friend posed a question to me last night. I have my own opinion and I
> gave it to him. I would be interested in hearing what other scientists
> might think on the matter:
>> I will change the details a little just to protect the innocent.
>> The experiment being conducted measure the effect of a particular drug on
> the levels of certain liver enzymes. The levels of the enzymes will be
> quantitated by western blotting.
>> 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??
>>>> The answer to me seems pretty obvious in either scenario, but I'd like to
> hear other opinions too.
>> John
>>hines at pharm.med.upenn.edu
