Date of Award

2005

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Animal Science

Advisor

Charles R. Wallace

Second Committee Member

Martin R. Stokes

Third Committee Member

Charles E. Moody

Abstract

A new technique has been developed for the purification of bovine placental lactogen (bPL) from an ammonium sulfate preparation, using an immunomagnetic separation process. This alternative procedure is used in cell and molecular biology to separate various types of proteins, cells and nucleic acids due to its advantages over the traditional methods. The first step of the bPL purification system was to isolate antibody (Ab) against bPL, called sheep anti-rabbit antibody. A two milliliter column of Immobilized Protein G, elution buffer (pH 2.8) and binding buffer (pH 5.0) were used during Ab purification process. The Ab activity was monitored by using a radioimmunoassay (RIA) prior to coupling to a biomag, which consists of a suspension of magnetic particles coated with iron oxide to provide primarily amine groups. These groups allow for covalent attaching to proteins or ligands with maintaining the biological activity. A suspension of magnetic iron particles conjugated with sheep anti-rab bit IgG was used in the experiment. The coupling efficiency was 80% determined by measuring protein concentration at 280nm. The next step was purification of endogenous bPL from bovine placental homogenate, which had been prepared by an ammonium sulfate (A.S.) precipitation. One gram of dried A.S. dissolved in 40 ml Tris HCI buffer (1OmM pH 7.5) was centrifuged at 10,000 x g for 30 min. The supernatant was collected and mixed with 5 ml biomag. The particles were recovered with a magnet and the supernatant was discarded. This was followed by washing two times with Tris buffer (IOrnM pH 7.5). Finally, bPL was eluted from the biomag by adding 10 ml glycine (0.1M pH 3.2). Sucrose was then added to the glycine mixture to prevent protein aggregation and stored in the refrigerator for analysis. At each step of the purification process, the protein content of the sample was estimated by measuring the absorbance of light at 280 nanometers with a spectrophotometer. The Bradford assay was also utilized for further examining of protein level in the sample. The presence of bPL in the sample was determined by RIA. The purified bPL fraction was concentrated by lyophilization and run on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine how pure the bPL is. The purity level of the bPL was 25%, which is higher than results obtained in some previous studies using a similar starting material preparation. This result, however, is still not much greater than obtained with previous conventional bPL purification approaches, and other studies using the immunomagnetic separation techniques to isolate the target biomolecules. Thus further purification steps are required. The purification system described in this study, at present, cannot offer an efficient protocol for the isolation of bPL. The developed method, nevertheless, is simple and rapid as only a one-purification step is involved. This approach has also reached a moderate sensitivity, which may perhaps be refined to enhance the optimization. Additional investigations, regarding the conditions of elution buffers and the size of magnetic particles, may need to be taken into consideration to obtain a better result.

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