Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed

<p>Abstract</p> <p>Background</p> <p>Flow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail.</p> <p>Findings</p> <p>To address this question, we analyzed the effect of RNAlater on fluorescence intensity of GFP, YFP, DsRed and small fluorescent molecules attached to secondary antibodies (Cy2 and Texas-Red) when used in flow cytometry. FACS results were confirmed with fluorescence microscopy. Our results showed that exposure of YFP and GFP containing cells to RNAlater reduces the intensity of their fluorescence to such an extent that separation of such labeled cells is difficult if not impossible. In contrast, signals from DsRed2, Cy2 and Texas-Red were not affected by RNAlater treatment. In addition, the background fluorescence and clumping of dissociated cells are altered by RNAlater treatment.</p> <p>Conclusions</p> <p>When considering gene expression studies using cell sorting with RNAlater, DsRed is the fluorescent protein of choice while GFP/YFP have severe limitations because of their reduced fluorescence. It is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties (e.g., clumping) of the cells before considering its use in cell sorting.</p

Multigrid solution of the nonlinear Poisson-Boltzmann equation and calculation of titration curves.

Although knowledge of the pKa values and charge states of individual residues is critical to understanding the role of electrostatic effects in protein structure and function, calculating these quantities is challenging because of the sensitivity of these parameters to the position and distribution of charges. Values for many different proteins which agree well with experimental results have been obtained with modified Tanford-Kirkwood theory in which the protein is modeled as a sphere (reviewed in Ref. 1); however, convergence is more difficult to achieve with finite difference methods, in which the protein is mapped onto a grid and derivatives of the potential function are calculated as differences between the values of the function at grid points (reviewed in Ref. 6). Multigrid methods, in which the size of the grid is varied from fine to coarse in several cycles, decrease computational time, increase rates of convergence, and improve agreement with experiment. Both the accuracy and computational advantage of the multigrid approach increase with grid size, because the time required to achieve a solution increases slowly with grid size. We have implemented a multigrid procedure for solving the nonlinear Poisson-Boltzmann equation, and, using lysozyme as a test case, compared calculations for several crystal forms, different refinement procedures, and different charge assignment schemes. The root mean square difference between calculated and experimental pKa values for the crystal structure which yields best agreement with experiment (1LZT) is 1.1 pH units, with the differences in calculated and experimental pK values being less than 0.6 pH units for 16 out of 21 residues. The calculated titration curves of several residues are biphasic

Ligation alters the pathway of urea-induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase.

We have examined the pathway and energetics of urea-induced dissociation and unfolding of the catalytic trimer (c3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N-(phosphonacetyl)-L-Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl- and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the absence of active-site ligands and in the presence of ATP, c3 dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity. Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for delta Gdissociation at 7.5 degrees C of about 7-10 kcal m-1 per interface. delta Gunfolding of dissociated chains when modeled as a 2-state process is estimated to be very small, on the order of -2 kcal m-1. The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1-state swelling process. In the presence of the ligands CP and PALA, and in the presence of Cl-, c3 dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand