western blot quantitation

arturo.galvani at itner.pharmacia.se arturo.galvani at itner.pharmacia.se
Wed Jun 21 02:21:13 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
     
     ______________________________________________________________________
     
     Hi William,
     
     I've attempted quantitation from western blots developed using 
     enhanced chemiluminesence (ECL).  Using this system with various 
     antigens, and I suspect that the same will hold true for other 
     detection systems, I found that within a certain range of antigen 
     concentration, the response (measured as integration of band optical 
     density) was linear, but that linearity was lost when non-optimal 
     (either low or high) amounts of antigen protein were loaded. I guess 
     that the optimal range of antigen amount will depend on a number of 
     factors such as the antibodies you are using, the detection system, 
     etc. 
     Substrates which colour the filter, such as NBT are not so hot for 
     quantitation, however, because you cannot easily do densitometry. 
     There are substrates available for both horse-radish peroxidase and 
     alkaline phosphatase which generate luminescent signals, giving 
     autoradiogram-type hard copies which can then be analysed by 
     densitometry.  I suggest you use one of these substrates, and load 
     serial dilutions of your antigen onto the gel to determine the range 
     of linear response in the system you are using.
     
     
     Arturo Galvani
     Pharmacia Biopharmaceuticals Milan
     (speaking for myself)




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