Homemade Chemiluminescent Substrates?

Keld Sorensen KeldS at uic.edu
Fri May 30 07:38:55 EST 1997

Thomas Cameron wrote:
> Company kits end up costing $3-5 per western blot which is 90% of the
> cost of the whole procedure.

Say you use 10 ml of a 1:500 dilution of a primary antibody that cost
you $1,000/mg, then the antibody cost is $20 per blot. If you had to use
say a 1:250 dilution (weak affinity antibody) then the cost would be $40
per blot!!

Then there is the cost for the membrane  - it can easily be a couple of
$ per blot depending on how you buy, down to maybe less than $1 if you
buy rolls (assuming a 10 cm x 10 cm blot) - but it is the same no matter
where you get the substrate (obviously).

You may use a cheaper Ab, or you may have one you made yourself, but if
$3 is 90% of your cost, then you must use a cheap membrane, so that the
antibody cost less than say 10 cents per 10 ml (this leaves 20 cents for
the membrane) - again assuming the 1:500 dilution, you must have paid
less than $5 per mg for your antibody - that is very cheap! If homemade
antibody was used, the cost of one rabbit alone would bring the price
way over that. Maybe somebody donated the Ab to you, but then they had
the cost.

The cost of the secondary antibody is usually inconsequential  - say you
use 1:40,000 dilution of a conjugate that cost $100 per mg,  then 10 ml
is costing you 2-3 cents!!

The cost of wash buffers etc is also trivial (and unavoidable).

This calculation obviously does not negate your thought that less
expensive can be helpful - it is just that the size of the savings may
be a lesser percentage of the overall cost than you thought - when you
compare substrates, it is important to compare $/ml of substrate used
per blot and take into account possible differences in signal intensity,
or dilution of antibody required for a given signal. Quite a complex
task by the way, and perhaps best done with an experiment (i.e. "what
dilution of the substrate can I get away with with a given HRP conjugate
concentration?" or "what dilution of the primary antibody can I get away
with given the substrate used?")

Hope this helps.

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