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.

Quality Control of Bacterial Extracellular Vesicles with Total Protein Content Assay, Nanoparticles Tracking Analysis, and Capillary Electrophoresis

Abstract: Extracellular vesicles (EVs) were isolated from Pectobacterium zantedeschiae culturing media using direct ultracentrifugation (UC), iodixanol cushion ultracentrifugation (ICUC), and iodixanol density gradient ultracentrifugation (IDGUC) techniques. The isolates were characterized with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A satisfactory correlation (R2 > 0.94) between quantitative results obtained with BCA, NTA and CE was achieved only for isolates obtained with the IDGUC. The correlation between protein content and CE was proved to be related to the isolates’ purity. The NTA was found unable to provide reliable information on EVs quantity in samples isolated with UC and ICUC, due to the co-isolated particulate impurities. Moreover, the work reports polysaccharides, used as culturing media components, as a potential source of bias of quantitation with total protein content assay and NTA. The study demonstrates the advantageous selectivity of CE in quality control of EVs and its ability to differentiate subpopulations of EVs of Pectobacterium.

Characterization of protein binding to a nitrocellulose membrane

Abstract: Proteins bound to a nitrocellulose (NC) membrane tightly and quantitatively until the NC membrane was saturated with the proteins. The binding of proteins to the NC membrane was characterized by using twenty proteins having different molecular weight and isoelectric point as follows: 1) hydrophobic interactions between the protein and the NC membrane matrices may play a major role in the protein binding, and sugars, amino acids, DNA, nucleotides, neutral salts, or glycerol in the protein solution did not interfere with the protein binding. 2) The number of the protein molecule bound to the NC membrane was in the range from 1.13 to 1.98nmol/cm2 except for a few small and strongly charged proteins. 3) Non-ionic detergents such as Tween 20, Triton X-100, and Nonidet P-40 strongly interfered with the protein binding to the NC membrane in a concentration dependent manner. 4) Proteins could be extracted from the NC membrane with high yield by using a low concentration of the non-ionic detergent. These enables us to assay the proteins with high sensitivity and reproducibility. Furthermore, it may be possible to determine the amino acid composition and sequence with small amount of the proteins after the assay.

Comparisons of protein extraction procedures and quantification methods for the proteomic analysis of Gram-positive Paenibacillus sp. strain D9

Abstract: Four protein extraction methods and three protein quantification techniques were compared with Paenibacillus sp. whole cells. Proteins were extracted using conventional cell disruption techniques encompassing: sonication and glass bead vortexing, as well as BugBuster Master Mix extraction and Total Protein Kit extraction. The Bradford assay, Folin-Lowry assay and UV absorbance at 280 nm were used for protein quantification methods. Differences in protein profiles were examined by 2D-PAGE and subsequently analysed using PDQuest Advanced 2D Analysis software. All extraction methods revealed proteins over broad molecular weight range. UV absorbance at 280 nm using the NanoDrop™1000 and the Bradford assay yielded best quantification results. Rapid and effective disruption and quantification of Paenibacillus sp. strain D9 cells was successfully achieved using the combination of Total Protein Extraction Kit-UV280 followed by BugBuster Master-UV280.