Mouse igg antibodies

SDS/PAGE was carried out as described in [25 (link)] with our modification. The separating gel with 7% polyacrylamide was used [25 (link)]. The stacking gel contained 4% polyacrylamide and was prepared as described in [26 (link)]. Western blotting was performed as described in [27 (link)]. 9D10 anti-vertebrate striated muscle mouse monoclonal antibody was used to detect titin. Mouse IgG antibodies (Sigma–Aldrich) were used as secondary antibodies conjugated with horseradish peroxidase.

The antibody binding to the Arf GAP with the SH3 Domain, Ankyrin Repeat and PH Domain (ASAP1) protein was tested using the recombinant ASAP1, (OriGene Inc., Rockville, MD, USA) using an immunoblot assay. The blots were probed with primary antibodies (human anti-peptide antibodies or mouse control monoclonal antibody) followed by either peroxidase-conjugated anti-human immunoglobulin antibodies or mouse IgG antibodies (Sigma) (10 μg per millilitre). A monoclonal antibody against ASAP1 was purchased from OriGene and used as a positive control. A monoclonal antibody directed against beta-actin was used as an equal loading control (Abcam).
The blots were developed using chemiluminescence according to the manufacturer’s instructions (Thermo Scientific).

Cells were lysed in lysis buffer (50 mM Tris-HCl, with 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100, pH 7.4) supplemented with a complete protease inhibitor cocktail (Thermo 78438) and phosphatase inhibitor (Thermo 78427), followed by centrifugation at 4 °C for 5 min. The supernatant was then precleared with mouse IgG agarose beads (Sigma A0919) at 4 °C for 4 h and subsequently mixed with washed agarose beads conjugated with anti-Flag (Sigma A2220), anti-HA (Thermo 26182), anti-Myc (Sigma A7470, anti-AKT (Cell Signaling Technology 3653), or mouse IgG antibodies (Sigma A0919) at 4 C° overnight. Immunocomplexes were washed extensively 3 times with washing buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). The immunoprecipitates were eluted with 2× sodium dodecyl sulfate (SDS) and then subjected to immunoblotting analysis.
For transfection experiments, 6 × 10⁸ cells were seeded in 10 cm dishes and transfected with 5 µg of each plasmid using Lipofectamine 2000 (Thermo 11668019) for 48 h. Cells were then treated and lysed as described above.