Volume 101, Issue 11, Pages 2807-2815 (December 2011) Analyzing the Homeostasis of Signaling Proteins by a Combination of Western Blot and Fluorescence Correlation Spectroscopy  Yi-Da Chung, Michael D. Sinzinger, Petra Bovee-Geurts, Marina Krause, Sip Dinkla, Irma Joosten, Werner J. Koopman, Merel J.W. Adjobo-Hermans, Roland Brock  Biophysical Journal  Volume 101, Issue 11, Pages 2807-2815 (December 2011) DOI: 10.1016/j.bpj.2011.09.058 Copyright © 2011 Biophysical Society Terms and Conditions

Figure 1 Determination of cellular protein concentrations using FCS for calibration of WB. Lysates of cells transiently expressing the protein of interest fused to an FP are analyzed in parallel by FCS and WB. With knowledge of the confocal detection volume Veff (Section S3), the molecule number N obtained by FCS is converted into a concentration of FP (Cf) in the lysate. This concentration is then used to calculate the endogenous protein concentration through determination of the ratio (Re/f) between the endogenous and recombinant fluorescent fusion protein, as obtained from WB analysis. Biophysical Journal 2011 101, 2807-2815DOI: (10.1016/j.bpj.2011.09.058) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 2 Determination of the fraction of fluorescing fusion proteins. (A) Scheme depicting the protocol for determining the fraction of fluorescing fusion proteins. Concentrations of FPs (Cfluo) are derived from the number of fluorescent molecules (Nfluo), as provided by FCS, divided by the confocal detection volume (Veff). The concentration of total FP (Ctotal) is determined by using standard quantitative WB of known amounts of purified FPs as a standard and probing with an antibody against FP. Band intensities (I) for purified FPs on WB versus the respective molecule numbers (N) in the samples are plotted as a standard curve for determining unknown samples. The fluorescent fraction of FP (F) is given by the ratio Cfluo/Ctotal. (B) WB of FPs probed with an antibody against monomeric DsRed for detection of mCherry. Different amounts of cell lysates or recombinant proteins were loaded into the individual lanes (lanes 1, 3, 5, 7, 9, and 14: purified mCherry; lanes 2 and 12: mCh-GADS; lanes 4 and 11: mCh-SHP1; lanes 6 and 8: transiently expressed mCherry; lanes 10 and 13: mCh-GRB2). (C) Comparison of the endogenous GRB2 concentration determined by the conventional quantitative WB method (black bar) and the FCS-based method without (white bar) and with (gray bars) correction of the fraction of fluorescing proteins, without (left) and with (right) propagation of the error for fluorescing fractions of FPs. Biophysical Journal 2011 101, 2807-2815DOI: (10.1016/j.bpj.2011.09.058) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 3 Determination of endogenous GRB2. (A) WB of lysates of mCherry and mCherry-GRB2-transfected Jurkat cells probed with an anti-GRB2 antibody. Samples containing different amounts of lysate were loaded in a randomized fashion to compensate for drift in the gel. Lanes 2, 5, and 7 contain various volumes of lysates of mCherry-transfected cells. Lanes 1, 3, 4, and 6 contain various volumes of lysates of mCh-GRB2-transfected cells. The arrowhead in the upper panel indicates mCh-GRB2, and the one in the lower panel indicates the endogenous GRB2. (B) Linear regression analysis of background-corrected intensities of endogenous GRB2 (solid squares) and mCh-GRB2 (solid triangles) plotted versus cell numbers. (C) Autocorrelation functions of mCh-GRB2 in crude cell lysates. The global fit to the autocorrelation function is indicated as a solid black curve. Average numbers of molecules were derived from the amplitude of fitted autocorrelation curves. Biophysical Journal 2011 101, 2807-2815DOI: (10.1016/j.bpj.2011.09.058) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 4 Intracellular protein distribution. GRB2 (A and F), GADS (B and G), SHP1 (C and H), and LAT (D and I) were either expressed as fluorescent fusion proteins (A–D) or detected by immunofluorescence (F–I). (E and J) Autofluorescence controls were either not transfected (E) or incubated only with secondary antibody (J). Scale bar indicates 5 μm. Biophysical Journal 2011 101, 2807-2815DOI: (10.1016/j.bpj.2011.09.058) Copyright © 2011 Biophysical Society Terms and Conditions