Bradford protein assay

About: Bradford protein assay is a research topic. Over the lifetime, 635 publications have been published within this topic receiving 239107 citations.

A comparison of Lowry, Bradford and Smith protein assays using different protein standards and protein isolated from the marine diatom Thalassiosira pseudonana

Abstract: Two spectrophotometric assays for protein commonly used in marine research (Coomassie stain, “Bradford”; alkaline copper, “Lowry”) and a more recent assay which has not been applied in this field (bicinchoninic acid, “Smith”) were compared for homogenates of the marine diatom Thalassiosira pseudonona using bovine serum albumin (BSA) as a standard. When homogenates were prepared by precipitating protein with trichloroacetic acid (TCA) and redissolving in 1 N NaOH, the protein content estimated by the Lowry and Smith assays agreed closely, but was consistently 20% higher than that indicated by the Bradford assay. To determine if this difference was due to the choice of a protein standard, protein from T. pseudonana was purified and compared to BSA, bovine gamma-globulin (BGG), and casein. The reactivity of the purified protein (expressed as the slope of the absorbance vs protein concentration curve) did not differ between cultures grown at high or low irradiance. For the Smith and Bradford assays the reactivity of BSA was not significantly different from algal protein, but for the Lowry assay, algal protein was significantly higher in reactivity than BSA. BGG was not significantly different in reactivity from algal protein for the Lowry and Smith assays, but BGG gave significantly lower absorbances than algal protein in the Bradford assay. These results suggest that BSA is a suitable standard for algal protein in the Bradford assays, while BGG is preferable for the Lowry assay. Either protein standard could be used for the Smith assay. Differences in purified algal protein reactivity compared to BSA could not account for the differences among the assays, nor could interference by chlorophyll a. Precipitating protein with TCA prior to analyses gave lower protein than direct analyses of homogenates for the Lowry and Smith assays, but no differences were found for the Bradford assay. As a result, the Lowry and Smith assays indicated up to 60% greater protein than the Bradford if TCA precipitation was not performed. This may be due to removal of free amino acids and small peptides which are less reactive in the Bradford assay. The 20% higher protein found in the Lowry or Smith vs Bradford assays may be due to different assay sensitivity to small peptides or other compounds which are precipitated along with proteins by TCA. Although the Smith assay is substantially simpler to perform than the Lowry, there appear to be no quantitative differences in the results. It remains unclear which spectrophotometric assay is most accurate, but the Bradford assay is faster and simpler, and is less likely to be affected by non-protein compounds found in marine phytoplankton.

Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps

Abstract: Strips of horticultural film (16–32 cm2) were used to trap extraradical hyphae emanating from roots of sudangrass [Sorghum sudanense (Piper) Staph] enclosed in 40-μm mesh bags and colonized by Gigaspora rosea FL 224-1, Glomus intraradices EY 113/114, or Glomus caledonium UK 301-1. Strips of film were placed at opposite sides of 17–21 replicate sand culture pots for each isolate and were removed after 12–14 weeks of plant growth. To extract glomalin, a strip was cut into small pieces and submerged in 2 ml of 20 mM citrate, pH 7.0 and then autoclaved for 60 min. A quantitative enzyme-linked immunosorbent assay (ELISA) detected 0.005–0.04 μg glomalin in the volume of extract tested. The Bradford protein assay detected 1.25–5 μg of protein in the volume of extract tested. Both assays gave results ranging from 5–40 μg glomalin/cm2 of film. Protein assay values were correlated with ELISA values (r=0.6091, P≤0.001, n=118). Analysis of variance indicated that isolates differed in Bradford protein values (P=0.001), but not ELISA values (P=0.154). Spatial variability of glomalin deposition ca. 7 cm from roots on opposite sides of pots was indicated by significant paired T tests (P<0.05) for protein values for each of the three isolates and ELISA for two isolates. These results indicate that hyphal traps, Bradford protein assay and ELISA are useful to assess hyphal activity over a growing season.

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

Abstract: Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.