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Journal of Undergraduate Research

Keywords

molecular targets, antimicrobial chemokines, CCL25, CCL28

College

Life Sciences

Department

Microbiology and Molecular Biology

Abstract

For this project, I hypothesized that the amino acid composition of the C terminus of chemokine proteins allows some chemokines to bind to different sites on the bacterial cell wall and that the chemokines with a strong positive charge (CCL25 and CCL28) will bind to the same target, while CCL27, which has no positively charged C terminus region, will have a different binding site. In order to study the binding properties of each chemokine, I began by performing binding assays. In these assays, approximately 105 colony-forming units of exponentially growing bacteria were incubated on ice with the appropriate chemokine. This allowed the chemokine to bind to the bacteria and resulted in low levels of bacterial lysis. After thirty minutes, any unbound chemokine was removed by washing in a PBS/calf serum wash buffer. The bacteria were then incubated with a second chemokine. Any unbound chemokine was again washed away. Next, the cells were incubated with an anti-chemokine biotinylated antibody specific to the second chemokine and any unbound antibody was washed away. Finally, an avidin-conjugated fluorochrome was added to the cells. The percentage of stained cells, as well as the intensity of staining on each cell, was read by flow cytometry. By comparing these results to the control set, in which no blocking antibody was added (omission of the first chemokine in the experiment described above), these assays should have allowed me to determine if the binding of the first chemokine was blocking the binding of the second chemokine.

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