4.4. Validating and characterizing fluorescent protein-tagged cytoskeletal proteins

Validating genomic tag insertion at the target locus can be done using a PCR screening method. Use isolated genomic DNA from transformants and untransformed cells as templates for the PCR reaction, and compare fragment sizes amplified by a primer pair that hybridizes up- and downstream of the tagging locus. Sequencing should then be carried out to verify that tagged genes do not carry any mutations.

Initial steps to confirm successful expression of tagged proteins in transformed cells can be performed through visual inspection. Prepare cells for short-term imaging as described below and screen several colonies for consistent fluorescent protein expression.

We recommend using Western blot analysis to study expression of tagged proteins. Benefits of protein expression monitoring include 1) comparing the expression level of tagged protein to that of its native form; 2) troubleshooting protein expression when the levels fall below the threshold of detection by microscopy; and 3) identifying degradation products that may hinder proper localization and function.

It is important to evaluate the localization and functions of tagged constructs and confirm that these are preserved, when knowledge of the gene of interest is available. Tagging proteins with fluorescent proteins may alter their function and/or localizations by disrupting native protein-protein interactions, or by inducing oligomerization through the fluorescent protein. Evaluating whether function is preserved in fluorescent protein-tagged strains can be done by examining their cell growth rates and loss of any characterized phenotypes of the gene of interest. Fluorescent protein-tagged strains with compromised protein function will exhibit similar phenotypes to cells bearing mutations in that gene. For a positive indicator of function, a plasmid-borne version of the fluorescent protein-tagged protein can be used to rescue a wild-type phenotype in yeast cells with a genomic deletion of the gene of interest (Note 6). After confirming proper localization and preserved function, strains can be used for live-cell analysis. If, on the other hand, target protein localization and/or function is found to be compromised by a fluorescent protein tag, an alternative approach is to fuse the target protein to a smaller epitope tag such as myc or HA and visualize the protein in fixed cells using immunofluorescence.