Electric Field Gradient Focusing-UV Detection for Protein Analysis Electric Field Gradient Focusing-UV Detection for Protein Analysis
Using fiber-based EFGF with UV detection, a concentration factor as high as 15,000 and a concentration limit of detection as low as 30 pM were achieved using bovine serum albumin as a model protein. I also demonstrated the potential of using fiber-based EFGF for quantitative protein analysis. Simultaneous desalting and protein concentration as well as online concentration of ferritin and simultaneous removal of albumin from a sample matrix were also performed using this fiber-based EFGF system.
In the approach of utilizing hydrogel-based EFGF, online concentration of amyloglucosidase indicated a concentration limit of detection of approximately 20 ng/mL (200 pM) from a sample volume of 100 micro-L. Both voltage-controlled and flow-controlled elution methods were demonstrated using a 3-component protein mixture. Concentration of human á1-acid glycoprotein with simultaneous removal of human serum albumin was also described.
A tandem EFGF system, which integrates fiber-based and hydrogel-based EFGF sections, was also investigated to selectively concentrate and separate proteins in a mixture. By carefully controlling the voltages applied to both sections, charged analytes with high mobilities were trapped in the fiber-based section, analytes with intermediate mobilities in the hydrogel-based section, and analytes with low mobilities not at all. A 3-way switching valve was incorporated in the system to purge the analytes with high mobilities periodically. Selective concentration and separation of myoglobin from a mixture were performed using the tandem EFGF system.
Based on the experimental results described in this dissertation, EFGF shows potential for selective isolation, concentration, and quantitation of trace analytes such as proteins in biomedical samples.
School:Brigham Young University
School Location:USA - Utah
Source Type:Master's Thesis
Keywords:electric field gradient focusing protein analysis desalting selective isolation and concentration of proteins tandem efgf
Date of Publication:04/26/2006