sodium-dodecyl-sulfate and bicinchoninic-acid

Fluorescent silica nanoparticle (NP) labels are of great interest in biomedical diagnostics, however, when used in bioassays under physiological conditions they rapidly agglomerate and precipitate from solution leading to high levels of non-specific binding. In this work, using size and zeta-potential data obtained from Dynamic and Electrophoretic Light Scattering analysis, the improvement in colloidal stability of silica NPs under physiological conditions was correlated with an increase in the concentration of three additives: (1) a protein, bovine serum albumin (BSA); (2) a neutral surfactant, Tween 20®; and (3) a charged surfactant, sodium dodecyl sulfate (SDS). The number of BSA molecules present in the NP corona at each concentration was calculated using UV-Vis spectroscopy and a bicinchoninic acid protein assay (BCA). The optimal concentration of each additive was also effective in stabilizing antibody labeled fluorescent nanoparticles (αNPs) under physiological conditions. Using a fourth additive, trehalose, the colloidal stability of αNPs after freeze-drying and long-term storage also significantly improved. Both as-prepared and freeze-dried αNPs were tested in a standard fluorescence immunoassay for the detection of human IgG. The as-prepared assay showed a higher sensitivity at low concentration and a lower limit of detection when compared to a free dye assay. Assays performed with freeze dried αNPs after 4 and 22 days also showed good reproducibility.

Topics: Colloids; Fluorescent Dyes; Freeze Drying; Humans; Immunoassay; Immunoglobulin G; Light; Nanoparticles; Particle Size; Polysorbates; Quinolines; Reproducibility of Results; Scattering, Radiation; Serum Albumin, Bovine; Silicon Dioxide; Sodium Dodecyl Sulfate; Spectrophotometry, Ultraviolet; Surface-Active Agents

Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations Sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and Sonication-BCA_P combinations.

Topics: Biochemistry; Cell Culture Techniques; Cells, Cultured; Chloroform; Escherichia coli; Hydroxides; Metabolism; Microbiological Techniques; Models, Statistical; Models, Theoretical; Potassium Compounds; Proteins; Quinolines; Sodium Dodecyl Sulfate; Spectrophotometry, Ultraviolet; Toluene

A new solid-phase, fluorescence-based protein assay was developed that quantifies proteins in the presence of detergents, urea and reducing agents (one-dimensional sodium dodecyl sulfate (1-D SDS) lysis buffers and urea isoelectric focusing (IEF) buffers). A specially designed 96-well microplate facilitates application of protein samples to the assay paper and allows easy quantitation of samples using fluorescence microplate readers (top or bottom reading format). Alternatively, stained membranes may be directly scanned using a variety of different laser or charge-coupled device (CCD)-based imaging devices with UV or visible imaging capabilities. Since protein is specifically bound to the membrane, contaminants are readily washed away, avoiding interference with the protein measurement. The protein assay has a dynamic range extending from 10 ng to 5 microg of protein per microliter and requires only 1 microL of sample, which is ideal for samples destined for electrophoresis. The protein-to-protein variability of staining of ten different proteins was determined to be comparable with that of the bicinchoninic acid (BCA) and Lowry assays (16%). Additionally, the quality of the assay according to Z-factor analyses is excellent.

Topics: Animals; Detergents; Electrophoresis; Fluorescent Dyes; Humans; Isoelectric Focusing; Proteins; Quinolines; Reducing Agents; Sodium Dodecyl Sulfate; Urea

The bicinchoninic acid (BCA) assay method for the determination of protein has been investigated for its utility in measuring the protein content of plasma lipoproteins. Although other methods, principally those based on the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275) have been extensively used for this purpose, the tolerance of the BCA method to many commonly encountered detergents and buffers offers a definite advantage over the Lowry-based methods. In this study, lipoprotein protein values obtained by the BCA method were compared to a standard modification of the Lowry et al. procedure since this assay forms the basis of much of the relevant literature. The standard BCA assay was found to overestimate the protein content of very low density lipoprotein by approximately 70% and low density lipoprotein by approximately 30%; high density lipoprotein values compared favorably. Overestimations by the BCA assay paralleled the relative phospholipid content of the lipoprotein fractions. This apparent lipid effect was eliminated by the addition of 2% sodium dodecyl sulfate to samples prior to the analysis. In the presence of this detergent, BCA assay measurements for these three lipoprotein fractions were 97, 90, and 98%, respectively, of the reference assay values.

Topics: Humans; Lipoproteins; Phospholipids; Proteins; Quinolines; Sodium Dodecyl Sulfate; Spectrophotometry

Topics: Glycine; HEPES; Mercaptoethanol; Microwaves; Octoxynol; Polyethylene Glycols; Proteins; Quinolines; Sodium Dodecyl Sulfate; Time Factors; Tromethamine