Horseradish peroxidase conjugated anti mouse igg secondary antibody

Mouse brain tissue and cells were lysed in NP-40 lysis buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40), and protein concentration in the cell lysate was quantified using a Bradford protein assay (Bio-Rad). Protein samples were incubated at 100 °C for 5 min and electrophoresed on 10% SDS-polyacrylamide gels. Proteins were transferred onto PVDF membranes (GE Healthcare Life sciences). Membranes were blocked in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h. Membranes were then incubated at 4 °C with a primary antibody mouse anti-Eps8 (BD Transduction) and mouse anti-GAPDH (Millipore) diluted in 5% nonfat milk overnight (1:500 and 1:10,000, respectively) then washed several times with TBS-T. After 1 h incubation with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:10,000) (GE Healthcare Life sciences), membranes were washed with TBS-T and protein bands on the membrane were detected using an ECL-Plus immunoblotting chemiluminescence system (GE Healthcare Life sciences). Membranes were imaged using ImageQuant LAS 500TM camera (GE Healthcare Life sciences).

Cell-free synthesized DDSP domains were diluted 20-fold, injected into a 96-well MaxiSorp plate (Nunc, Rochester, NY, USA), and incubated overnight at 4 °C. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBST), the plate was blocked with TBST containing 5% skimmed milk for 1 h at room temperature. Next, the plate was incubated with an anti-DYKDDDDK tag monoclonal antibody (012–22384, FUJIFILM Wako Pure Chemical, Osaka, Japan) or anti-biotin antibody (A4541, Sigma-Aldrich) diluted 1:2000 and 1:1000, respectively, in TBST containing 5% skimmed milk for 1 h at room temperature. Thereafter, the plate was washed three times with TBST and incubated with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (GE Healthcare, Chicago, IL, USA) diluted in TBST containing 5% skimmed milk for 1 h at room temperature. Finally, 50 µL of tetramethylbenzidine liquid substrate (Sigma-Aldrich) was injected into the plate and incubated for 15–30 min at room temperature. The reaction was terminated by injecting the same volume of 1 M HCl. Absorbance at 450 nm was measured using a SpectraMAX M3 plate reader (Molecular Devices, San Jose, CA, USA).