[Protein-analysis] Re: quantifying western blots
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Mon Jun 19 07:39:34 EST 2006
Joerg Schaber wrote:
> Thanks for your reply.
> Of course, I should have mentioned that I do not want to determine
> absolute protein quantities but only relative quantities for each gel,
> by scaling the maximal value on each gel to one, for instance. Just to
> get some numbers.
Yes, but you have to be aware that there is a non-linear relationship
between those numbers and the protein amount.
> When I understood you correctly you propose the following:
> 1) Quantify the band for a specific lane (e.g. by counting black pixels
> in a rectangle around the band).
> 2) define the background for this lane (e.g. by counting black pixels in
> a rectangle placed next to the one above but in the same lane)
> 3) Substract the the two values.
> 4) do this for each lane.
The shape of the band changes from lane to lane. So I'd confirm by
Western that there is only one protein reacting with the antibody, but
for quantitation I'd use dot blots. The sample is passed over a
protein-binding membrane in a 96-well vacuum manifold, that way it is
distributed over a defined area. Millipore for example offers 96-well
plates with membrane bottom for such applications.
The blot is then developed like a western (blocking, primary antibody,
washing, secondary, washing). If you want good linearity in
quantitation, use a 96-well chemoluminescence counter and a
chemoluminescent detection kit for HRP or AP-labeled secondary
antibodies. This type of assay is linear over 5-6 orders of magnitude,
the common colorimetric or photographic detection not even for one order
Some of the modern detection systems for fluorescence may offer similar
linearity, but I haven't layed my hands on one of those yet.
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