PMC:4829102 / 29341-30947 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4829102","sourcedb":"PMC","sourceid":"4829102","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4829102","text":"Immunoprecipitation experiments\nLysates for co-immunoprecipitation experiments were prepared as follows; cells were washed twice in PBS and then lysed in IP buffer (100 mM NaCl, 0.2% Igepal CA-630, 1 mM MgCl2, 10% glycerol, 5 mM NaF, 50 mM Tris-HCl, pH 7.5), supplemented with complete EDTA-free protease inhibitor cocktail (Roche) and 25 U ml−1 Benzonase (Novagen). After Benzonase digestion, the NaCl and EDTA concentrations were adjusted to 200 mM and 2 mM, respectively, and lysates cleared by centrifugation (16,000 × g for 25 min). Lysates were then incubated with 20 μl of GFP-Trap agarose beads (ChromoTek) blocked with 5% BSA in IP lysis buffer for 1h at 4 °C in the case of GFP trap IPs or with 20 μl of anti-FLAG M2 affinity gel in the case of FLAG IPs for 2 hours with end-to-end mixing at 4 °C. Complexes were washed extensively in IP buffer (including 200mM NaCl and 2 mM EDTA) before elution. In the case of GFP trap IPs, beads were resuspended in 2X SDS sample buffer and boiled for 3 min before centrifugation at 5000 × g for 5 min. The resultant supernatant fraction was retained as the eluate. In the case of FLAG IPs, beads were incubated for 30 min with gentle agitation at 4 °C in IP buffer supplemented with 400 μM 3x FLAG peptide (Sigma) followed by centrifugation at 5000 × g for 5 min. The resultant supernatant fraction was collected as eluate.\nFor mass spectrometry analyses eluates from immunoprecipitation experiments were analysed by the Mass Spectrometry Laboratory (IBB PAS, Warsaw, Poland) using the Thermo Orbitrap Velos system and protein hits were identified by MASCOT.","divisions":[{"label":"Title","span":{"begin":0,"end":31}}],"tracks":[]}