Showing papers on "Bradford protein assay published in 1984"

Protein, cholesterol, acid phosphatase and aspartate aminotransaminase in the seminal plasma of turkeys (Meleagris gallopavo) producing normal white or abnormal yellow semen.

Abstract: Turkeys which produce yellow semen have abnormal ductuli efferentes’ epithelial morphology, with b!ebbing of cytoplasmic material into the ductal lumen. This could possibly increase the activity or concentration of seminal plasma components. In the present study, seminal plasma from 270 Large White breeder turkeys was evaluated for protein and cholesterol concentrations and the activities of acid phosphatase and asparate aminotransaminase. In a separate experiment, protein concentrations of turkey seminal plasma were estimated by biuret or Bradford methods. Bradford estimates were 46.6% less than those obtained with the biuret assay, using bovine serum albumin as the standard. Estimates of seminal plasma protein concentration in the main study were obtained using the Bradford method, and should be adjusted accordingly when compared with other studies using the biuret technique. Abnormal yellow seminal plasma, compared to normal white seminal plasma, had elevated levels of total protein and cholesterol and increased activities of acid phosphatase and aspartate aminotransasninase. Overall means were: 14.3 mg/mI, 38.9 mg/dl, 232.6 lU/mI, 81.0 lU/mI, respectively. Correlation coefficients for cholesterol concentration, acid phosphatase and aminotransaminase activity with protein concentration were +0.65, 0.70 and 0.50 (P<0.0001), respectively. Specific activities of both enzymes showed a significant reduction as seminal plasma protein increased, indicating a disproportionate increase in proteins other than these enzymes in yellow seminal plasma.

Protein assay by silver binding

Abstract: A novel, inexpensive, highly sensitive and quantitative assay for measuring protein in solution based on the capacity of protein to bind silver is described. In this procedure, protein samples are first treated with glutaraldehyde and then exposed to ammoniacal silver. After a specified time, the reaction is terminated by the addition of sodium thiosulfate and the optical density measured at 420 nm. The useful range of the assay for the majority of standard proteins tested lies between 15 and 2000 ng. This represents a 100-fold increase in sensitivity over the Coomassie Brilliant Blue dye binding procedure. There is little or no interference from carbohydrates, non-ionic detergents or ethanol. Pretreatment of protein samples with Bio-Gel P-2 to remove salts, thiol agents, EDTA and SDS, makes this procedure compatible with most commonly used buffers.

Anomalous Behavior of H1 Histone and Polylysine in the Bradford Assay for Protein

Abstract: In the assay for proteins described by Bradford,' the reagent is an aqueous solution of 0.01% (wt/vol) Coomassie brilliant blue G-250 containing 4.75% (wt/vol) ethanol and 8.5% (wtlvol) phosphoric acid to which is added 0.02 volumes of protein solution. Proteins cause an increase in the absorbance of light with a shift in an absorption maximum from 645 to about 595 nm. Histones were reported to give a color yield that was very similar to that obtained with bovine serum albumin (BSA).* We confirmed this observation with a mixture of total calf thymus histones. However, we found that H 1 histones from either calf thymus or rat liver were much less effective in shifting the peak wavelength and increasing light absorption (FIGURE 1). In contrast, using the assay procedure of Lowry et ul.,' we saw a close correspondence between the results with BSA and HI histone. To further characterize the anomalous behavior of H I histone in the Bradford assay, we examined chemically related substances. The effect of polylysine was similar to that of H 1 histone whereas the H2B and H4 histones gave spectral changes similar to those of BSA. Lysine monomer and spermine had a negligible effect on the absorption spectrum of the dye. The response to HI histone and polylysine cannot simply be attributed to the basic nature of these polypeptides, because protamines, which are arginine-rich polypeptides, gave a greater color yield than BSA. In addition, the presence of 0.05 N NaOH did not decrease the color yield with BSA after correction for the appropriate control. Read and Northcote' presented evidence that variation in response to different proteins in the Bradford assay could be diminished by decreasing the concentration of phosphoric acid. We found this to be true for BSA and H I histone. Under the standard assay conditions, the absorption at 595 nm in the presence of H1 histone was less than 20% of that with BSA. If the phosphoric acid concentration was halved, the absorption in the presence of BSA was decreased and the absorption with H1 histone was increased (TABLE 1). The linearity of the chromogenic response with protein concentration was less for HI histone than for BSA. An alternative approach to equalizing sensitivity to various proteins in the Bradford assay is the addition of sodium dodecyl sulfate (SDS). In agreement with Macart and Gerbaut; we found that SDS decreased the sensitivity of the assay for BSA. The color yield with H I histone was increased with SDS concentrations up to 0.1 % (wt/vol) but decreased at higher concentrations. The color yield with BSA and H1 histone in the Bradford assay could be made approximately equal by decreasing the phosphoric acid concentration to 6% or by making the protein solution 0.1% with respect to SDS. A disadvantage of the former' modification was an increase in the absorbance of the reagent control. A disadvantage of the latter modification was a lower sensitivity. Although the color yields with H1