Analysis of the interferences in quantitation of a site-specifically PEGylated exendin-4 analog by the Bradford method.

Protein modification has been found to affect the estimation of protein concentration in some of the traditional dye-based absorbance measurements. In this work, a distinct reduction in A595 was observed during the quantitation of a PEGylated exendin-4 analogue (Ex4C) by the Bradford method and the PEGylation process was found to interfere with the measurement. Lys(12), Arg(20), and Lys(27) were further proved to be the major amino acids that functioned as dye-binding sites. The shielding effect produced by the large polymer was demonstrated to depend on the length of PEG that was used for modification.

Comparison of two methods of tear sampling for protein quantification by Bradford method / Comparação de dois métodos de coleta de lágrima para quantificação proteica pelo método de Bradford

The aim of this study was to compare two methods of tear sampling for protein quantification. Tear samples were collected from 29 healthy dogs (58 eyes) using Schirmer tear test (STT) strip and microcapillary tubes. The samples were frozen at -80ºC and analyzed by the Bradford method. Results were analyzed by Student's t test. The average protein concentration and standard deviation from tears collected with microcapillary tube were 4.45mg/mL ±0.35 and 4,52mg/mL ±0.29 for right and left eyes respectively. The average protein concentration and standard deviation from tears collected with Schirmer Tear Test (STT) strip were and 54.5mg/mL ±0.63 and 54.15mg/mL ±0.65 to right and left eyes respectively. Statistically significant differences (p<0.001) were found between the methods. In the conditions in which this study was conducted, the average protein concentration obtained with the Bradford test from tear samples obtained by Schirmer Tear Test (STT) strip showed values higher than those obtained with microcapillary tube. It is important that concentration of tear protein pattern values should be analyzed according the method used to collect tear samples.

Compararam-se dois métodos de coleta de lágrima para quantificação proteica, utilizando-se 58 olhos de 29 cães hígidos. As amostras foram coletadas utilizando-se fitas de teste da lágrima de Schirmer (Schirmer tear test - STT) e tubos microcapilares. Após obtidas, as amostras foram congeladas a -80º C e posteriormente analisadas pelo método de Bradford. Os resultados foram analisados pelo teste T de Student. A média da concentração proteica e desvio padrão das amostras obtidas com microcapilar foi de 4,45mg/mL ±0,35 e 4,52mg/mL ±0,29 para olhos direito e esquerdo, respectivamente. Para as amostras obtidas com STT os resultados foram 4,45mg/mL ±0,35 and 4,52mg/mL ±0,29 para olhos direito e esquerdo, respectivamente. Diferenças estatisticamente significativas (p<0,001) foram encontradas entre os dois métodos. Nas condições em que o trabalho foi conduzido, a média da concentração proteica pelo método de Bradford das amostras obtidas através das tiras do STT foi superior à obtida com o tubo microcapilar. Os valores padrão da concentração protéica da lágrima obtida pelo método de Bradford devem ser analisados considerando-se o método de coleta da lágrima, uma vez que este interfere significativamente nos resultados obtidos.

Interpretation of biological and mechanical variations between the Lowry versus Bradford method for protein quantification.

BACKGROUND: The identification of differences in protein expression resulting from methodical variations is an essential component to the interpretation of true, biologically significant results. AIMS: We used the Lowry and Bradford methods- two most commonly used methods for protein quantification, to assess whether differential protein expressions are a result of true biological or methodical variations. MATERIAL #ENTITYSTARTX00026; METHODS: Differential protein expression patterns was assessed by western blot following protein quantification by the Lowry and Bradford methods. RESULTS: We have observed significant variations in protein concentrations following assessment with the Lowry versus Bradford methods, using identical samples. Greater variations in protein concentration readings were observed over time and in samples with higher concentrations, with the Bradford method. Identical samples quantified using both methods yielded significantly different expression patterns on Western blot. CONCLUSIONS: We show for the first time that methodical variations observed in these protein assay techniques, can potentially translate into differential protein expression patterns, that can be falsely taken to be biologically significant. Our study therefore highlights the pivotal need to carefully consider methodical approaches to protein quantification in techniques that report quantitative differences.