Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots

Int J Mol Sci. 2022 Jan 21;23(3):1195. doi: 10.3390/ijms23031195.


G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This results in the recruitment of arrestins, leading to desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffolding partners. GRKs' versatility is also reflected by their diverse roles in pathological conditions such as cancer, malaria, Parkinson's-, cardiovascular-, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6) in Western blot analysis. We identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross-reactivity. Hence, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained Western blot results. Utilizing the most-suitable antibodies, we established the Western blot-based, cost-effective simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in nine commonly used cell lines, revealing differential isoform expression.

Keywords: GRK2; GRK3; GRK5; GRK6; Western blot; antibody specificity; protein quantification.

MeSH terms

  • Animals
  • Antibodies / immunology*
  • Blotting, Western / methods*
  • CHO Cells
  • Cricetulus
  • G-Protein-Coupled Receptor Kinases / analysis*
  • G-Protein-Coupled Receptor Kinases / immunology
  • G-Protein-Coupled Receptor Kinases / metabolism*
  • HEK293 Cells
  • Humans
  • Isoenzymes
  • Mice
  • NIH 3T3 Cells
  • Phosphorylation
  • Rats
  • Signal Transduction


  • Antibodies
  • Isoenzymes
  • G-Protein-Coupled Receptor Kinases