To each tube 120 ml alkaline lysis buffer 2 (0.2 M NaOH, 1% (w/v) SDS) was added and the mixture was shaken and incubated on ice for 10–20 min. Then 210 ml of cold alkaline lysis buffer 3 (4 M sodium acetate, 2 N acetic acid) was added and the mixture was shaken to mix ...
After discarding the supernatants, protein was eluted from the beads by addition of 20 µl 2 × SDS Laemmli buffer and heating at 95 °C for 10 min. 14 µl of each sample were loaded on 12% SDS-polyacrylamide gels for Western blot analysis. RT-qPCR analysis Total RNA ...
Pierce™ Protein G Magnetic Beads (20 μL, Thermo Fisher Scientific Fisher) were washed three times with washing buffer (50 mM Tris–Cl (pH 7.4), 0.5 M NaCl, 0.05% v/v TWEEN® 20) and incubated with 2 μ
For co-immunoprecipitation, Pierce™ Protein G Magnetic Beads (20 μL, Thermo Fisher Scientific Fisher) were washed three times with washing buffer (50 mM Tris–Cl (pH 7.4), 0.5 M NaCl, 0.05% v/v TWEEN® 20) and incubated with 2 μg of rabbit anti-LRP10 (Sino Biological, ...
For each mRNA assessment, q-RTPCR analyses were repeated 3 times as previously done (Anderson et al., 2015, Aras et al., 2019, Ramadori et al., 2019). Immunoblotting and Immunoprecipitation Immunoblots were performed according to standard procedures in RIPA buffer (150 mM NaCl, 10 mM ...
After centrifugation at 400g for 5 min, cells were resuspended in fluorescently labeled LDLR antibody or TFR antibody diluted in 100 μl blocking buffer and incubated for 1 h in the dark at 4°C. Cells were then washed three times with ice-cold PBS, resuspended in 150 μl cold PBS for...
(GH Healthcare cat BR-1000-14) to a level of approx. 2000 RU (resonance units). Purified CH2 domain mutants are dialyzed against 10 mM Hepes (pH 6.0), 150 mM NaCl (running buffer), diluted in the same buffer to a range of concentrations (10 μM-0.625 μM) and passed over the ...
times with wash buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA). The proteins were eluted from beads by boiling in 50 µl 2 × SDS sample buffer and separated on 8% SDS-PAGE gels. Gel blots were analyzed using anti-MBP (NEB, 1:5000...
Marrow was spun and incubated with red blood cell (RBC) lysis buffer (Sigma) to remove red blood cells. A single cell suspension was prepared by passing the cells through a 70 μm cell strainer (Corning). They were then plated in 10 cm petri dishes in complete DMEM (Dulbecco’s ...
Next, the lysate with primary antibody was incubated with 50 μl of sepharose A beads per IP reaction (Invitrogen, cat no. 101041) pre-equilibriated with lysis buffer for 2 h at 4 °C. After centrifugation, beads were then washed three times with IP buffer for 10 min each. SDS loading...