Anti mouse igg veriblot for ip secondary antibody

Two days after transfection, HEK-293T cells were washed once with pre-cooled PBS and lysed on ice for 30 min in lysis buffer (10 mM Hepes, pH 7.6, 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1:1000 protease inhibitor). For immunoprecipitation, 2.4 μg anti-GFP monoclonal antibody (Roche) and 30 μl of protein G-Sepharose beads (GE Healthcare Bio-Sciences) or 30 μl anti-FLAG M2 agarose from mouse (Sigma A2220) were added to each lysate. Mixtures were incubated overnight at 4°C with rotation; the supernatant was removed, and protein beads were washed three times using 0.5 m lysis buffer. Cells were fixed for immunofluorescence staining in PBS 3.7% formaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. After being washed with PBS 3 times, cells were incubated in DMEM+0.02% Azide with the appropriate primary antibodies, then incubated with Alexa-405/488/594 conjugated second antibodies (Invitrogen). For Western blot analysis, samples were subjected to electrophoresis in 8% or 10% SDS-polyacrylamide gels and immunoblotted using the polyclonal antibody specific to anti-FLAG antibody, anti-GFP antibody, anti-cherry antibody, anti-SSRP1 antibody (PA 5-22186, rabbit, Thermo), or anti-SPT16 antibody (H-300, sc-28734, Santa Cruz). Anti-mouse IgG VeriBlot for IP secondary antibody (HRP) (ab131368, Abcam) was used.