Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

Abstract

Sphingosine kinase 2 (SK2) is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis . Therefore, well in vivo validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs). Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout ( ) MEFs. Our results demonstrate that the Proteintech Sphk2 anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

“…To determine if this band was in fact SK2, the ECM Biosciences anti-SK2 antibody was then used to immunoprecipitate SK2 from HEK293 lysates treated with either scrambled control or SK2-directed siRNA. SK2 knockdown consistently resulted in reduced intensity of the band enriched by this antibody ( Figure 2C; Dataset 2 19 ), confirming that the ECM Biosciences anti-SK2 antibody can selectively IP endogenous SK2.…”

“…The ECM Biosciences anti-SK2 antibody did result in consistently observable staining in HeLa and HEK293 cells, which was substantially reduced upon knockdown of SK2 ( Figure 3A and B; Dataset 3 20 ). Hence, in these cells the ECM Biosciences antibody was able to selectively detect SK2 by IF.…”

“…The Proteintech anti-SK2 antibody is suggested by the manufacturer to be useful for IP, whereas to our knowledge the ECM Biosciences anti-SK2 antibody has not been previously tested for use in this application. Initially, using lysates from HEK293 cells, we found that the Proteintech anti-SK2 antibody was sometimes able to IP a band at the correct size for SK2 ( Figure 2A); however this was not consistent with each experimental repeat and other proteins were also immunoprecipitated to a varying extent by this antibody that were not present in the IgG isotype control ( Dataset 2 19 ).…”

“…To determine the selectivity of the anti-SK2 antibodies, we performed IB analyses using two human cell lines (HEK293 and HeLa) that had been treated with either scrambled control or SK2-directed siRNA, as well as WT and Sphk2 -/- MEFs. The Proteintech anti-SK2 antibody detected a single prominent band at the correct molecular weight for SK2 (~65 kDa), which was decreased or absent in the knockdown and knockout lines ( Figure 1A; Dataset 1 18 ). Some faint non-specific bands were also detected in both the WT and Sphk2 -/- MEF lysates by this antibody, which were not observed in the human cell lines.…”

“…Conversely, the ECM Biosciences anti-SK2 antibody was able to consistently and cleanly IP a protein of the same size as SK2 from cell lysates, with almost no non-specific bands observed ( Figure 2B; Dataset 2 19 ). The protein immunoprecipitated by the ECM Biosciences antibody was considerably enriched from the cell lysate and was strongly detectable by this antibody, which was unable to detect SK2 in the lysate input sample, consistent with Figure 1B.…”