Purification of Actin Capping Protein Antibodies using Affinity Chromatography

1. Sixxy Sivongxay and Dr. Marilyn Hart Department of Biological Sciences, Minnesota State University-Mankato Introduction Antibodies are an effective tool used in many biological applications. In previous studies in the laboratory of Dr. Marilyn Hart, an antibody was generated to be used as a marker for actin capping protein in cells and tissues. Actin capping protein modulates the activity of actin monomers and filaments. The generated antibodies are a complex mixture of specific and nonspecific antibodies. Therefore, we are using affinity chromatography to purify our antibody of interest from a heterogeneous group of molecules. The initial antibody mixture will be passed through an affinity column to allow binding, a wash buffer will be applied to remove unbound nonspecific antibodies and the elution buffer will subsequently be applied to release the specific antibodies. Fractions will be collected and their protein concentrations determined. A fraction with an increased concentration will be identified suggesting that the antibodies were purified. • Methodology • 1. The affinity column and the solutions (0.2M Glycine, 0.1M NaHCO3, TTBS Buffer, and 10X PBS Buffer) have been prepared prior to the start of the experiment. • 2. The initial antibody mixture will be passed through the column to allow binding. • 3. Glycine and NaHCO3 buffers will be applied to the column three times to remove any residual unbound antibodies. • 4. The column will be balanced in TTBS Buffer + 0.5M NaCl. • 5. The serum will be prepared by adding 1mL 10X TTBS + 0.55mL 4M NaCl to 10mL sample of serum. This mixture will then be centrifuged to remove any precipitates and debris. • 6. Elution process. The column will be washed extensively with TTBS + NaCl until A280 is less than 0.02 to remove unbound antibodies. • 7. 10mL fractions of the washes will be collected and their protein concentrations determined using Optical Density (Spectrophotometry) and the column will be drained. • 8. A single fraction with an elevated concentration will be identified, suggesting that the alpha 2 IgG antibodies have been purified. Results The Alpha 2 IgG antibodies in the rabbit will be purified from the complex mixture of antibodies using affinity chromatography. The affinity column, solutions, and , the serum have already been prepared. We have completed the background and literature review. The experiment is ongoing. Purification of Actin Capping Protein Antibodies using Affinity Chromatography Conclusion The Alpha 2 IgG antibodies in the rabbit will be purified from the complex mixture of antibodies using affinity chromatography. Future Studies Use the purified Alpha 2 IgG antibodies in Western Blot Studies and Fluorescent Staining. Acknowledgements This research project was partially funded through the McNair Achievement Program. Special thanks to Dr. Marilyn Hart, my faculty mentor, and the Minnesota State University-Mankato Department of Biological Sciences. Phylogenetic analysis of the alpha subunit of actin capping protein indicates that although alpha 1 and alpha 2 are highly conserved (same branch), unique sequences define membership to the alpha 1 and alpha 2 subfamilies. Scientific Question Can the Alpha 2 IgG antibodies in the rabbit serum be purified from the complex mixture of antibodies? Initial complex mixture of antibodies will be passed through the affinity column to allow binding. Buffers will be applied to remove unbound nonspecific antibodies. Elution buffers will be applied to release the specific antibodies.