Chapter 6 In-Cell NMR in Mammalian Cells: Part 3 Beata Bekei, Honor May Rose, Michaela Herzig, Heike Stephanowitz, Eberhard Krause, and Philipp Selenko Abstract Irrespective of how isotope-labeled proteins are delivered into mammalian cells, laboratory routines are needed to assess the quality of the resulting in-cell NMR samples. These include methods to evaluate overall cell viability, protein transduction efficiency, intracellular protein concentration, localization, and stability. In addition, quality control experiments to assess protein leakage from manipulated cells are of particular importance for in-cell NMR experiments. The purpose of this chapter is to outline qualitative and quantitative methods to determine general biological properties of in-cell NMR samples in order to ensure the highest possible standards for in-cell NMR studies. Key words: Trypan blue, Flow cytometry (FCM), Fluorescence live cell imaging, Immunofluores- cence microscopy, Western blotting, Quantitative mass spectrometry, Quality control 1. Introduction In the previous chapters we have outlined a collection of methods to deliver isotope-labeled IDPs into amphibian-, and cultured mammalian cells. While different protocols can be employed to prepare in-cell NMR samples, generic quality control experiments to verify the overall fitness of the obtained in-cell NMR specimens are of general importance. Questions concerning cell viability, pro- tein delivery efficiency, as well as intracellular protein localization and stability need to be addressed in order to critically assess the biological relevance of in-cell NMR results. In the following, we will outline a compendium of stringent quality control experiments that should additionally be performed on every in-cell NMR sam- ple. Most of the suggested experiments follow standard laboratory routines in cell biology. Therefore, we will only focus on those aspects of the protocols that are of particular importance for in- cell NMR experiments. Vladimir N. Uversky and A. Keith Dunker (eds.), Intrinsically Disordered Protein Analysis: Volume 1, Methods and Experimental Tools, Methods in Molecular Biology, vol. 895, DOI 10.1007/978-1-61779-927-3_6, # Springer Science+Business Media, LLC 2012 67