Abstract
Cell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individual Escherichia coli cells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is ≥80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.
Highlights
Cell biology increasingly relies on quantitative microscopic labelling methods that provide strong fluorescent signals but are well vetted in terms of detection efficiency and specificity
Bacterial strains containing a cytoplasmic HaloTag protein expressed from a medium copy-number plasmid under the control of an arabinose inducible promoter were grown in a low auto-fluorescence medium to mid-exponential phase
HaloTag expression was induced by adding 1% arabinose for 1 hour, HTL-TMR was subsequently added at 5 μM final concentration and the cells were incubated for an additional one hour at 37 °C. We chose this concentration of HTL-TMR to ensure complete labelling of all HaloTag proteins
Summary
Cell biology increasingly relies on quantitative microscopic labelling methods that provide strong fluorescent signals but are well vetted in terms of detection efficiency and specificity. To test whether Halo-TMR labelling could be used to quantify variation in protein levels, we measured the average fluorescence intensity of cells expressing the HaloTag protein in the presence of various arabinose concentrations. These results indicate that the HaloTag labelling can be used to quantify protein levels in single cells with the same sensitivity as FP-based methods.
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