[Histonet] HPLC and Protein extraction

From:Brian Dias

Hi Everyone,
    Have just embarked upon HPLC analysis for 5-HT and 5-HIAA in lizards. 
Got some HPLC data that looked quite good (5-HT and 5-HIAA levels in fmol 
being different in pharmacologically manipulated animals). But when i 
normalized the data to amount of protein in the pellet, my results vanished 
due to the differences in protein levels in my samples (which were all over 
the place).
1.I figure that if i take the same number of punches per region per animal, 
my protein levels should not vary drastically within a region. Is that a 
wise assumption to make?

2. While performing the HPLC, i suspend my tissue punches in perchloric 
acid and spin it down. Does anyone know of a protocol i might look into to 
extract protein from this pellet. I'm presently using Sigma's Mammalian 
Cell Lysis kit and then using a protein assay by Pierce Biotech?

Any input will be appreciated.


    Brian George Dias

    Graduate Student (Crews lab)            Office Phone #: 512-475-6738
    The University of Texas at 
Austin       E-mail:   brian.dias@mail.utexas.edu
    Institute for Neuroscience                  Website: 
    Patterson 56
    1 University Station Stop C0930
    Austin, TX 78712

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