After protein lysates were collected, protein concentrations were determined via Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). For co-immunoprecipitation, Pierce™ Protein G Magnetic Beads (20 μL, Thermo Fisher Scientific Fisher) were washed three times with washing buffer (50 mM Tr...
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, ...
After completion of the gradient the refolded nucleosomes were dialysed two times against no-salt buffer (10 mM TRIS pH 7.5, 1 mM EDTA, 1 mM DTT) CD analysis of the H2A/B dimers. CD spectra were recorded using a Chirascan CD-spectrophotometer (Applied Photophysics). H2A/B dimers were ...
Cells were briefly spun, the blocking buffer was removed and the antibody cocktail was added as required (1:100 or 1:200 dilution in blocking buffer), and incubated overnight at 4 °C. The following day the cells were spun, rewashed three times with PBS-T, and Alexa-fluor fluorescent...
Baselines are adjusted to zero for all curves and double-referenced by subtracting a sensorgram of buffer injected over the coated surface from the experimental sensograms to give curves representing specific binding Curves are modeled assuming a simple 1:1 interaction to generate the equilibrium and...
After centrifugation, beads were then washed three times with IP buffer for 10 min each. SDS loading buffer was finally added to the beads and loaded on a SDS-PAGE gel after denaturation at 90 °C for 5 min. Input loaded on the gel was equivalent of 10 % of the total protein used ...
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...
Aberrant translation causes ribosome stalling, which leads to the ubiquitination of ribosomal proteins and induces ribosome-associated quality control. As part of this quality control process, the E3 ubiquitin ligase RNF10 monoubiquitinates ribosomal pro
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...
fruit samples were treated with ABA for indicated times before sampling.dY1H assay showing the binding of FvABF3 to the promoter fragment ofFvALKBH10B. The empty pGADT7 was used as the negative control. AbA Aureobasidin.eEMSA reveals that FvABF3 directly binds to the ABREcis-acing elements ...