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The first two on-chip reactions were discarded in order to stabilize the chip condition.
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For all the cells, a single microsphere was formed in each microwell under all the chip conditions, and such microsphere configurations remained throughout the culture period.
Furthermore, the microsphere diameters of each type of cell were strongly positively correlated with the microwell diameters of the chips, suggesting that microsphere diameter can be factitiously controlled by using different chip conditions.
We have optimized the ChIP conditions and identified an AR antibody that yielded high specificity in the ChIP experiments (Figure S1).
In addition, appropriate controls including positive control (RNA polymerase antibody), negative control (mouse IgG) and internal control (input) were established to optimize the ChIP conditions.
Furthermore, we confirmed that our ChIP conditions were specific enough that we did not get enrichment of the sequence 1200bp downstream of the Hsp26 promoter (Figure 1B).
ChIP conditions were similar to those used previously, except chromatin was liberated by micrococcal nuclease.
For TRIM33/PU.1 ChIP in B-ALL, 50 million cells were used for each ChIP conditions with 5 μg antibody.
For histone modification ChIP experiments in B-ALL, 20 million cells were used for each ChIP conditions with 2 μg antibody.
Ideally, earlier studies will have identified genomic sites where the protein is known to bind, and these sites can be used to optimize the ChIP conditions.
Taken together, these results showed that the HCT116-3 6) cells and the ChIP conditions we established can be used to study the promoter occupancy profiles of p53 and p73.
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