Abstract
The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling. Positioning reporter molecules as close as possible to the target remains a challenge in primary cells or tissues from patients that cannot be easily genetically modified. Indirect immunolabeling introduces relatively large linkage errors, whereas site-specific and stoichiometric labeling of primary antibodies relies on elaborate chemistries. In this study, we developed a simple two-step protocol to site-specifically attach reporters such as fluorophores or DNA handles to several immunoglobulin G (IgG) antibodies from different animal species and benchmarked the performance of these conjugates for 3D STORM (stochastic optical reconstruction microscopy) and DNA-PAINT (point accumulation in nanoscale topography). Glutamine labeling was restricted to two sites per IgG and saturable by exploiting microbial transglutaminase after removal of N-linked glycans. Precision measurements of 3D microtubule labeling shell dimensions in cell lines and human platelets showed that linkage errors from primary and secondary antibodies did not add up. Monte Carlo simulations of a geometric microtubule-IgG model were in quantitative agreement with STORM results. The simulations revealed that the flexible hinge between Fab and Fc segments effectively randomized the direction of the secondary antibody, while the restricted binding orientation of the primary antibody’s Fab fragment accounted for most of the systematic offset between the reporter and α-tubulin. DNA-PAINT surprisingly yielded larger linkage errors than STORM, indicating unphysiological conformations of DNA-labeled IgGs. In summary, our cost-effective protocol for generating well-characterized primary IgG conjugates offers an easy route to precise SRM measurements in arbitrary fixed samples.
Highlights
The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling
These conditions are best met by genetic fusions of the target protein with a tag, like photoswitchable fluorescent proteins for PALM, or enzymes (SNAP, Halo) that mediate the conjugation with organic fluorophores or DNA handles for DNA-PAINT, respectively
Site-specific and quantitative functionalization of antibodies with fluorescent dyes or single-stranded DNA (ssDNA) with the help of microbial transglutaminase (mTG) and subsequent click reactions was highly consistent for the tested immunoglobulin G (IgG), as opposed to common NHS labeling, which varies more strongly from batch to batch and with the number of reactive lysine residues on different antibodies
Summary
Site-specific and quantitative functionalization of antibodies with fluorescent dyes or ssDNA with the help of mTG and subsequent click reactions was highly consistent for the tested IgGs, as opposed to common NHS labeling, which varies more strongly from batch to batch and with the number of reactive lysine residues on different antibodies. Similar functionalization results for a range of other compatible IgG subtypes and primary antibodies can be expected based on sequence analysis except mouse IgG2 and rabbit IgG (Figure 1 and Supplementary Table S1). Our precise determination of labeling shell dimensions based on averaged microtubule cross-sections and the modeling of antibody complex conformations allowed an unequivocal interpretation of linkage errors when using antibodies for SRM, summarized in Figures 2i and 3. The larger outer dimensions in DNA-PAINT compared to STORM require more straightened conformations of 1°+2° antibody complexes and even exceed the theoretical maximum epitope−reporter distance in 1° antibodies We speculate this could be related to partial loss of IgG quaternary structure upon DNA conjugation. Comparable p-values were obtained by comparing the fit results of individual microtubule cross-sections (Supplementary Figure S3) for two conditions by an unpaired t-test
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