The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence

Abstract

Moesin is a cytoskeletal adaptor protein, involved in the modification of the actin cytoskeleton, with relevance to Alzheimer’s Disease. Well characterized anti-Moesin antibodies would benefit the scientific community. In this study, we characterized ten commercial antibodies for Moesin in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence

“…Our standard protocol involved comparing readouts from wild-type (WT) and knockout (KO) cells. [11][12][13] The first step was to identify a cell line(s) that expressed sufficient levels of Profilin-1 to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million "TPM" + 1), which we found to be a suitable cut-off (Cancer Dependency Map Portal, RRID: SCR_017655).…”

The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence

“…Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells. [7][8][9] To identify a cell line that expresses adequate levels of Sequestosome-1 protein to provide sufficient signal to noise we examined public proteomics databases, namely PAXdb (RRID:SCR_018910) 10 and DepMap (RRID:SCR_017655). 11 U2OS was identified as a suitable cell line and thus U2OS was modified with CRISPR/Cas9 to knockout the corresponding SQSTM1 gene (Table 1).…”

“…For Western blot experiments, we resolved proteins from WT and SQSTM1 KO cell extracts and probed them side-byside with all antibodies in parallel 8,9 (Figure 1).…”

“…For immunoprecipitation experiments, we used the antibodies to immunopurify Sequestosome-1 from U2OS cell extracts. The performance of each antibody was evaluated by detecting the Sequestosome-1 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates 8,9 (Figure 2). For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.…”

“…Immunofluorescence was performed as described in our standard operating procedure. 8,9,12 U2OS WT and SQSTM1 KO were labelled with a green and a deep red fluorescence dye from Abcam (cat. number ab176735 and ab176736), respectively.…”