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A secondary biotinylated sheep anti-mouse antibody, diluted 1∶200 in TNB buffer, was incubated for 30 minutes and followed by the enhancement steps of the indirect TSA-kit.
Each sample in HDAC buffer was incubated for 12 h at 37 °C.
After washing, peroxidase-conjugated anti-mouse IgG (diluted 1 : 1000 in the blocking buffer) was incubated for 60 min at 37°C.
Samples were then washed three times for five minutes with PBS and the second antibody (POX-anti-rabbit, 1 50 in blocking buffer) was incubated for three hours at room temperature in a humid chamber.
After four washes with PBS-T 0.05 %, HRP-conjugated rabbit anti-human IgG (Roche Applied Science) diluted (1 5,000) in the blocking buffer was incubated for 1 hour at 37 °C under gentle agitation.
Afterwards 50 μL of 30 μg mL−1 complementary target in hybridisation buffer was incubated for different times and at varying temperatures to optimise the displacement of the mutated-HRP, followed by the detection with TMB in the same manner.
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Monocytes (1 × 106 cells∙mL− 1 in PGC buffer) were incubated for 10 min at 37 °C with test compounds or vehicle (0.025% ethanol [v·v− 1]) and then stimulated for 10 min with ionophore A23187 (2.5 μM).
After extensive washing 3 times serial dilutions of serum samples in ELISA buffer were incubated for 2 hours at room temperature.
One hundred µL of IgG at a concentration of 0.0205 g/L and diluted in blocking buffer were incubated for 90 min at 18°C.
Horseradish peroxidase-conjugated anti-mouse IgG antibodies (Promega, diluted 1∶7500) in 100 µl antibody buffer were incubated for 1h at 37°C.
Briefly, the cell lysates were collected in hypo-osmotic buffer after treatment with PBS (control) or IL-6 (20 ng/ml) for 20 min, and the cell lysates (5 µg), synthetic peptide CK2 substrate (RRRADDSDDDDD; 0.1 mM), and γ-32P-ATP in the assay dilution buffer were incubated for 10 min at 30°C.
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