This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measurement at 280 nm is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein. An alternate protocol uses absorbance at 205 nm to calculate the protein concentration. Both methods can be used to quantitate total protein in crude lysates and purified or partially purified protein. Use of a spectrofluorometer or a filter fluorometer to measure the intrinsic fluorescence emission of a sample solution is also described. The measurement is compared with the emissions from standard solutions to determine the concentration of purified protein. The Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to an unknown protein, is also presented, as is the Lowry method, which measures colorimetric reaction of tyrosyl residues in an unknown.

The success or failure of protein-centered projects can frequently be traced to the quality of the analytical procedures used to characterize the sample at different stages. Qualitative and quantitative analysis can aid in definition of the sample for the purpose of designing separations. A knowledge of the properties of the desired protein (e.g., whether it has a high aromatic amino acid content) can suggest methods of analysis that will help locate the desired protein in a complex mixture. Establishing the properties of an isolated protein creates benchmarks that future researchers can use to evaluate their protocols and final product. Accurate quantitation of the amount of protein at the beginning, middle, and end of a series of steps is the only valid way to evaluate the overall yield of a procedure. The observation of significant loss of protein, without a substantial increase in the purity of the desired protein, following a particular purification procedure would indicate that the procedure should be omitted or revised.Several spectroscopic procedures for characterizing protein samples are described in UNIT 10.1A. Measuring the absorbance of the aromatic amino acids in a protein at difference wavelengths yields a very useful measure of protein concentration. This is non-destructive and requires very little sample or time. A more qualitative, but much more sensitive, evaluation is provided by fluorescence spectroscopy. Quantitation of the amount of protein contained in a solution also can be conveniently accomplished using colorimetric methods. The Bradford and the Lowry methods are the most frequently used and reliable procedures. The method of choice is the Bradford method, which is easy and rapid to complete.Another approach is amino acid analysis-qualitative analysis to determine purity and quantitative analysis to provide concentrations-both of which are presented in UNIT 10.1B. Procedures are also given for calculating amino acid composition from primary analytical data. Significant advances that have improved the precision and sensitivity of amino acid analysis have reinvigorated this method, which had for some years been neglected.Supplement 35

Factors controlling proliferation of adrenocortical cells have been studied in Although several purified polypeptides have been reported to stimulate the proliferation of animal cells-in culture, a high degree of cell specificity has not been observed. Fibroblast growth factor (FGF) is mitogenic for fibroblast, adrenocortical, myoblast, smooth muscle, chondrocyte, vascular endothelial, granulosa, and luteal cells, whereas epidermal growth factor (EGF) is mitogenic for fibroblast, corneal epithelial, certain mammary epithelial, and granulosa cells (1). Several pituitary polypeptides long considered trophic for specific target cells in ivo either inhibit DNA synthesis when added directly to target cells in culture or have no effect on growth (2-6). Understanding of the mechanisms controlling organ-specific growth is, therefore, incomplete; interaction of several growth factors and hormones may be involved (7).A recently developed strain of bovine adrenocortical cells in culture has been used to study factors controlling proliferation of the adrenal cortical cell (8). FGF but not EGF stimulates proliferation of these cells, whereas corticotropin (ACTH) and prostaglandin El (PGE1), which stimulate cyclic AMP formation, inhibit proliferation (8, 9). Corticotropin and PGE1 both stimulate steroidogenesis and induce the steroid biosynthetic pathway. The present studies indicate that angiotensin II and III at low concentrations stimulate DNA synthesis as well as steroidogenesis in adrenocortical cells. The growth-stimulatory effect of angiotensin appears specific because several cell types, including cells reported to possess angiotensin II receptors, do not increase DNA synthesis with addition of angiotensin II (Schwarz/Mann) was routinely added to cultures 12 hr after addition of growth stimuli. Twelve hours later, medium was removed and 1 ml of a 1% aqueous solution of Triton X-100 was added. The cells were incubated with this solution for 5 min and the entire contents of the plate

This unit describes spectrophotometric methods for measuring the concentration of a sample protein in solution. These methods are most appropriate for purified proteins. Some food components may interfere (or absorb) at these wavelengths. In Basic Protocol 1, absorbance measured at 280 nm (A 280) is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein (a 280). In the Alternate Protocol, absorbance measured at 205 nm (A 205) is used to calculate the protein concentration. The A 280 and A 205 methods can be used to quantitate total protein in crude lysates and purified or partially purified protein. Both of these methods are simple and can be completed quickly. The A 280 method is the most commonly used. The A 205 method can detect lower concentrations of protein and is useful for dilute protein samples, but is more susceptible to interference by reagents in the protein sample than the A 280 method. Basic Protocol 2 uses a spectrofluorometer or a filter fluorometer to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from standard solutions to determine the sample concentration. The fluorescence emission method is used to quantitate purified protein. This simple method is useful for dilute protein samples and can be completed in a short amount of time.

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