Goat anti mouse igg conjugated with cy3

For immunoprecipitation, the spinal cord tissue was isolated from E12.5 C57Bl6 embryos and lysed in the RIPA-buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, Triton X-100 1 % (v/v), Na-Deoxycholat 0.5 % (w/v), all Sigma-Aldrich) with protease inhibitors: 1 mM phenylmethanesulfonylfluoride (PMSF) and 1 μg/ml Aprotinin, all Roche. 30 μl Protein A/G agarose slurry (Santa Cruz Biotechnology, Dallas, TX) was incubated with 2.5 μg goat anti-mouse IgG conjugated with Cy3 (Dianova, Hamburg, Germany) for 1 h on a rotating wheel in PBS, followed by incubation with 4 μg of mAb 11E2 mouse anti-LRP1 α-chain (Storck et al. 2016 (link)) or 4 μg of isotype control mAb actin (BD) for 2 h in 1 ml PBS/A. 1000 μg of total protein was incubated with pre-labeled agarose slurry in 500 μl of RIPA buffer overnight, washed gently 3 times. Before SDS-polyacrylamide gel electrophorese, samples were boiled for 5 min in loading buffer (60 mM Tris-HCl pH 6.8, 2.5% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.01 % bromophenol blue). 5750^LeX, 487^LeX and LRP1 α-chain epitopes were subsequently detected by Western blot analysis.