Abstract
Although fixed colocalization studies are useful to assess the sub-cellular localization or interaction of proteins in cells, some studies require live cells in order correlate colocalization to biological functions. The aim of this thesis is to assess colocalization at the cell-membrane with between the ion channel, Kv2.1 and lck-tagged GFP or TagRFP-T. Lck-GFP is shown to clearly label the plasma membrane of Chinese Hamster Ovary (CHO) cells and to colocalize with Kv2.1-RFP with a Manders’ coefficient of 0.994 ± 0.001, similar to its colocalization with the membrane lectin-conjugated organic dye Wheat Germ Agglutinin (WGA) Alexa-Fluor 488 (Manders’ coefficient = 0.986 ± 0.004). Next, I determined if the degree of colocalization between Kv2.1 and WGA was dependent on the fluorophore-conjugate, WGA tetramethylrhodamine (TMR) was studied and found to colocalize with Kv2.1-GFP with a lower Manders’ coefficient (0.774 ± 0.054). Next, a novel membrane marker fluorescent proteins, lck-TagRFP-T was generated and studied. Lck-TagRFP-T was found to colocalize with Kv2.1-GFP (Manders’ coefficient = 0.927 ± 0.037). Based on these results, it appears that lck-GFP and lck-TagRFP-T are superior plasma membrane makers than WGA conjugated to TMR. Finally, Threshold analysis of the data, a technique to normalize background fluorescence signals and autofluorescence artifacts suggested that Manders’ coefficients may not be accurate in all cases because of misalignment of the acquired images. In the future, I would titrate the amount of transfected DNA and apply protocols designed to test the alignment of the images prior to colocalization analysis.