![]() This can be alleviated in some cases as most proteins require interactions with other proteins to function properly and these can then act as limiting regulators for the system. The principal drawback of transient transfections is the degree of overexpression typically associated with it. Overexpression of proteins and fusion-proteins in particular, through transient transfections provide an easy way of introducing a mutant protein or study the distribution of a protein in cells. As pointed out in the literature, both immunofluorescence labeling, and transfection can skew the quantification of protein densities, a challenge that has stimulated recent method development in labeling protocols for super-resolution imaging. Prior to selection of imaging and analysis strategies to minimize possible misinterpretations of data quantification, protein labeling must be under essential control. Quantitative information of molecular coordinates can be used to extract protein densities, protein cluster sizes, and stoichiometry if applied accurately. In addition to improving resolution, as compared to conventional microscopy where no separation of labelled molecules occurs, super-resolution single-molecule localization imaging allows for quantitative analysis. pinpointing the location of the stochastically switching fluorescent molecules) is used to generate a reconstructed super-resolution image of the labelled sample. Finally, mathematical analysis of the detected fluorescence (i.e. To generate a full representation of the labeled sample, the stochastic switching and detection process is repeated many times (several thousand to tens of thousands frames are recorded). The spatio-temporally separate labeled entities are then used to map out cellular topology with increased resolving power. By labeling an investigated protein with a fluorescent reporter, which can be made to switch between a fluorescently detectable state (active, ON) and non-detectable state (non-active, OFF), separation in time and space of single molecules becomes possible. Super-resolution single-molecule localization microscopy, has in the last decade made it possible to visualize biological specimens on the nanoscale. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All STORM and PALM localization data are available at Dryad Digital Repository ( ).įunding: This work was supported by Swedish Research Council 2013-6041 to H.B., Swedish Research Council 2015-4198 to HJ.B., Swedish Research Council 2016-00968 to HJ.B., Swedish foundation for strategic research RIF14-0091 to H.B Märta och Gunnar V. Received: AugAccepted: MaPublished: April 25, 2018Ĭopyright: © 2018 Bernhem et al. Heyrovsky Institute of Physical Chemistry, CZECH REPUBLIC PLoS ONE 13(4):Įditor: Marek Cebecauer, J. Results show that after 41h transfection, the total plasma membrane density of Na,K-ATPase increased by 63% while the endogenous contribution was reduced by 16%.Ĭitation: Bernhem K, Blom H, Brismar H (2018) Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging. Quantification of plasma membrane protein density revealed a time dependent increase of Na,K-ATPase, but no increase in size of protein clusters. ![]() Through incorporation of an N-terminal hemagglutinin epitope to a mMaple3 fused Na,K-ATPase (α 1 isoform), we analyze the spatial and quantitative changes of plasma membrane Na,K-ATPase localization during competitive transient expression. In this study we investigate the use of STORM and PALM microscopy to quantitatively monitor endogenous and exogenous protein expression. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |