Comparing antibodies for the detection of ch-TOG/CKAP5 in mitotic HeLa cells by indirect immunofluorescence

Citation

Shelford, J. & Royle, S.J. (2020). Comparing antibodies for the detection of ch-TOG/CKAP5 in mitotic HeLa cells by indirect immunofluorescence. figshare doi: https://doi.org/10.6084/m9.figshare.13395554.v1

Description

We tested different commercial antibodies by fixing and staining HeLa cells, using MLN8237 to test for specificity. In untreated cells we should see chTOG on the mitotic spindle and poles, and in MLN8237-treated cells, chTOG should be mislocalised.

Cells were treated with 0.3 µM MLN8237 before fixing. PTEMF (20 mM Pipes, pH 6.8, 10 mM EGTA, 1 mM MgCl2, 0.2 % Triton X-100, and 4 % paraformaldehyde) or ice-cold methanol fix were used. Antibodies described in the table were used at the concentrations suggested diluted in PBS with 3% BSA. Cells were co-stained for alpha Tubulin (Mouse, Sigma T6074 clone B-5-1-2, 1:1000) or alpha Tubulin (Rabbit, Invitrogen, 1:2000), depending on the species of the chTOG antibody. Secondary antibodies were conjugated to Alexa488 (chTOG) or Alexa568 (tubulin). Imaging was on a Nikon spinning disk confocal system. Scale bar, 10 µm.