quantitation of western blots

Colin Rasmussen colin_rasmussen at darwin.biochem.ualberta.ca
Wed Jun 21 01:04:39 EST 1995


william at neuro.usc.edu (William Sun) wrote:
>Hello all,
>
>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.
>
>-William

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 
quantified.

Colin






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