Topic
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.
Papers published on a yearly basis
Papers
More filters
TL;DR: Results showed that X10 gave a strong band shift observed in Lanes 6 and 7 for both 200 ng and 400 ng elute 1 samples; however, there was no shift in the bands for the wild-type, positive control.
Abstract: The aims of this work were to: i) purify GST-fusion protein from bacterial cell extracts of Escherichia coli; ii) quantify the protein by SDS PAGE and Bradford assay; iii) determine protein-DNA interaction of the purified protein by Electrophoretic Mobility Shift Assay. Bacterial culture prepared by inoculation of a single E. coli colony that had a GST fusion protein (gst: six-X10 hd) constructed by ligation of the six-7-hd (X10) sequence into the BamHI and EcoRI sites of the vector pGEX-2T, grown overnight, was sonicated using Cole-Palmer Ultrasonic Homogenizer. Fusion protein was eluted from the beads with Tris-glutathione buffer (50 mM Tris [pH 8.1], 20 mM glutathione), which contained reduced Glutathione. SDS-PAGE was used to calculate the extracted bound protein. Total protein quantification was then estimated by the Bradford assay. Bovine Serum Albumin (BSA) absorbance values were used to plot the standard curve used to calculate the concentrations of the sample proteins. Nylon membrane was used for the electrophoretic transfer; membrane was cross linked and detected by Pierce’s Chemiluminescent Nucleic Acid Detection module. Results showed that X10 gave a strong band shift observed in Lanes 6 and 7 for both 200 ng and 400 ng elute 1 samples; however, there was no shift in the bands for the wild-type, positive control. The concentration of the elute 1 was obtained by the Bradford assay as 242.52 ng/μl and that of elute 2 was 106.30 ng/μl. Similarly, the result obtained by gel analysis was 300 ng/μl (0.3 μg/μl) and 150 ng/μl (0.15 μg/μl) for elutes 1 and 2 respectively.
01 Jan 2011
TL;DR: The aim of the experiment is to develop either new or modified analytical method for the analysis and detection of protein using newly introduced micro-plate reader equipped with Take3 microplate with the help of Gen-5 software.
Abstract: Analysis of protein depends particularly on protein concentration 1 . Protein concentration measurement is the most important part in the research work to conduct protein-related studies. Although there are many methods available for this purpose, each method has certain limitations. The aim of the experiment is to develop either new or modified analytical method for the analysis and detection of protein using newly introduced micro-plate reader equipped with Take3 microplate with the help of Gen-5 software. An ideal assay should be simple and easy to carry out. Another aspect to be taken into consideration are low inference, stability of measured components and low protein to protein variation 2 . This research work involves to measure protein concentration using various assays such as, absorbance at 280 nm, Bradford assay and BCA assay. Bradford and BCA assays are the most popular tools to quantify the protein sample. Bradford assay involves the measurement of absorbance at 595 nm 3 . BCA assay involves measurement of absorbance at 562 nm. BSA was used as protein standard because it is highly pure and inexpensive. The overall strategy is to develop a robust assay that uses the least amount of sample and test reagents . Successful completion of this work will aid protein researchers in quick identication and analysis of proteins.
TL;DR: It is shown that phenol extraction is an efficient and cost-effective method for analyzing protein kinase activity in polyacrylamide gels and compared the in gel renaturable activities of protein kinases extracted by phenol to those extracted by utilizing Triton or sodium dodecyl sulfate.
TL;DR: This chapter describes a method for the production and characterization of fungal acid proteases that maintains pH levels below those found in traditional zymographic systems to avoid the potential loss of activity that may occur in aspartic proteases under alkaline conditions.
Abstract: This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.
TL;DR: A semi-quantitative protein assay, taking advantage of the adsorption of proteins to hydrophobic surfaces, has been developed in this article, where the test solution was serially diluted in buffer drops which had been applied to a flat polystyrene surface.