Functional assays data normalization

The function of the protein assay is to enable data from different extracts to be normalized as amount of fos/myc/ lg protein. This controls for variation in the number of cells extracted and the efficiency of extraction and sample recovery. It is unimportant where cell suspensions or monolayers are to be compared under similar conditions, but rather important for comparison of, for example, myc levels in different tumor samples (12,13). 

FR expression may also be dependent on the histological classification of a tissue or cancer. For example, using a recently developed quantitative assay for measuring functional FR in cells and tissues, it was found that normal ovarian tissue and the mucinous form of ovarian cancer express very low levels of FR ( 1 pmol FR/ mg protein unpublished data, Endocyte, Inc., West Lafayette, IN). However, serous ovarian carcinomas express high amounts of FR, with the average expression level being 30 pmol FR/mg protein, or about 30-fold higher than that of the normal... 

The method use to normalize and express data should be decided preferably no later than at the end of the feasibility studies. Comparisons of results from day to day and among laboratories are most accurate when done using normalized data. For example, in ELISA systems, optical density (OD) values are absolute measurements that are influenced by ambient temperatures, test parameters, and photometric instrumentation. Therefore, results need to be calculated and expressed as a function of the reactivity of one or more serum control samples that are included in each run of the assay. 

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