Abstract
This protocol was developed to label proteins in bacterial cells with antibodies conjugated to a fluorophore for fluorescence microscopy imaging. The procedure is optimized to minimize morphological changes and also to minimize the amount of antibodies needed for the staining. The protocol can also be used with primary antibodies conjugated to a fluorophore. The method has been verified extensively (van der Ploeg et al., 2013), but it should be noted that one case in Caulobacter crescentus (Hocking et al., 2012) has been reported in which the localization of a protein changed upon fixation by formaldehyde/glutaraldehyde. However, the localization of the same protein in E. coli did not change.
Keywords: Protein localization, Fixation, Imaging, Fluorescence, Bacteria
Materials and Reagents
Equipment
Procedure
Notes
Fixation of the bacterial culture (either by formaldehyde/glutaraldehyde or by ethanol or methanol), which is essential for the immunolabelling procedure, gives an osmotic shock to the cells. The localization of membrane bound or membrane associated proteins or of cytosolic proteins is not affected by the osmotic shock. However, freely in the periplasm diffusing proteins can be shocked to the cell poles during fixation. Therefore, we do advise to verify the localization of periplasmic freely diffusing protein by analysis of the localization of fluorescent protein fusions to these proteins in combination with life imaging.
Recipes
Acknowledgments
The protocol described has been used in the following publications: Blaauwen et al. (1999); Aarsman et al. (2005); Potluri et al. ( 2010); Typas et al. (2010); Banzhaf et al. (2012); van der Ploeg et al. (2013) and Egan et al. (2014).
References
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