quantitation of western blots
colin_rasmussen at darwin.biochem.ualberta.ca
Wed Jun 21 01:04:39 EST 1995
william at neuro.usc.edu (William Sun) wrote:
>Does anyone out there know if the signal observed on a western blot is
>linear with the amount of protein loaded on a gel? Does this linearity
>depend on the detection method used? ie. radioactive vs chemical detection.
>I am using secondary antibody conjugated to alkaline phosphatase. I
>visulize the protein by adding NBT substrate. Thanks for any information.
Yes, up to the point where the filter becomes saturated with protein response is linear. As afr as
detection, I know that radioactive detection can be used to quantify where the linear range of the
response occurs, which if you think about it for a second must take into account the linear range of
the protein loaded and the film used for detection. A recent paper by Howarth & Stevenson (within
the past two years) worked this out for detection of a tight junction protein, ZO-1. It should be
applicable to other proteins as well. If you send me an email to remind me I can hunt the reference
for you from MedLine.
For enzymatic detection I frankly don't know if the production of precipitate can be accurately
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