Macs protein a g microbeads kit

Immunoprecipitations (IP) were performed using the µMACS Protein A/G MicroBeads Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Whole cell lysate of 5 × 10⁶ cells was mixed with cortactin 4F11 mouse monoclonal antibody (EMD Millipore; 1 µg per 500 µg protein lysate) and 50 µl Protein G MicroBeads, and incubated for 1 h on ice. For magnetic IP, the cell lysate was passed over a separation column placed in the magnetic field of a µMACS separator and washed four times with 200 µl cell lysis buffer. To conduct SDS-PAGE and immunoblotting analysis, the immune complex was eluted from the column using heated 1× SDS sample buffer. Rabbit antibodies (see Immunoblot analysis protocol) were used for immunoblotting to avoid the detection of any interfering IgG bands from the mouse antibody used during the IP protocol. Protein–protein interactions were depicted by immunoblotting, using antibodies specific to the putative co-immunoprecipitated targets. For mass spectrometric analysis, aliquots of the IP eluates were analyzed by Coomassie blue staining (Brilliant Blue R Concentrate; #B8647; Sigma) following SDS-PAGE (40 µl). Isolated bands were subjected to mass spectrometry (Core Unit Proteomics, Interdisciplinary Center for Clinical Research University of Münster, Germany).

Co‐immunoprecipitation (IP) experiments were performed using the µMACS Protein A/G MicroBeads Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions as previously described (Stock et al., 2019). Whole‐cell lysate of 5 × 10⁶ cells was mixed with anti‐FAK/anti‐Paxillin rabbit monoclonal antibodies (Cell Signaling; each 1 µg per 500 µg protein lysate) and 50 µL Protein G MicroBeads and incubated for 1 h on ice. For magnetic IP, cell lysate was passed over a separation column placed in the magnetic field of a µMACS separator and washed four times with 200 µL of µMACS cell lysis buffer. The immune complex was then eluted from the column using preheated 1 × SDS sample buffer. Mouse monoclonal antibodies were used for subsequent immunoblotting of interacting proteins as described above to avoid the detection of interfering IgG bands from the IP.