Quantification of total and phosphorylated STAT3 by calibrated western blotting

Abstract

Quantification of intracellular proteins is essential to understand signaling. Here, we describe quantification of the expression and phosphorylation of the transcription factor STAT3. We present isolation of total and phosphorylated STAT3 from cell lysates by immunoprecipitation, followed by SDS-PAGE and western blot together with known amounts of a calibrator protein that shares an epitope with the precipitated proteins. Finally, we explain how to relate the amount of precipitated protein to the amount of calibrator protein considering the efficiency of immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Dittrich et al. (2012)¹ and Reeh et al. (2019).².

Keywords: Antibody; Protein Biochemistry; Signal Transduction.

Graphical abstract

Figure 1

Schematic overview of the experimental set up to quantify STAT3 and (p)Y STAT3 The figure was generated using bioRender (https://biorender.com).

Figure 2

Quantification of STAT3 and p(Y) STAT3 by calibrated western blot (A) HEK293 cells stably expressing membrane-bound IL-6 receptor were either left untreated or stimulated with 20 ng/mL IL-6 for 15 min. Subsequently, cells were lysed and STAT3 and (p)Y STAT3 were precipitated using specific antibodies against STAT3 and (p)Y STAT3, respectively. Precipitates together with recombinant STAT3 calibrator proteins were analyzed by western blot using a specific antibody against STAT3. (B) Before (Pre) and after (Post) IP aliquots of the lysates were taken and analyzed by western blot using specific antibodies against STAT3, (p)Y STAT3, and HSC70. HSC70 serves as loading control. Figure 2 is modified from with permission of Royal Society of Chemistry.

Figure 3

Calibration curve Recombinant STAT3 calibrator proteins were analyzed by western blot using a specific antibody against STAT3 and a fluorophore-coupled secondary antibody (Figure 2A). Quantification was done by using Image Studio software (Table 1). The equation displayed is the result of a linear regression. R is the correlation coefficient. Figure 3 is modified from with permission of Royal Society of Chemistry.