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The resultant clonal cell lines were screened for PyLT expression by Western blotting (Figure 2A), and several clones with low basal and robust induced levels of PyLT (LT-5 and LT-6) were chosen for further study.
Individual clones were expanded and screened for physical linkage between pur and PTRE-HPRT by identifying clones with low frequency PTRE-HPRT inactivation (via TG selection) while retaining resistance to puromycin.
For all cell lines, clones with low expression levels were chosen for further analyses.
Several clones with low basal and high inducible psoriasin levels were isolated.
Cells with a high expression of tTA were transfected with the pBI-EGFP-psoriasin plasmid and clones with low background expression and homogeneous psoriasin induction were selected.
Colonies containing the BAC were screened for clones with low copy number integration and transfected with Flp to excise the selection cassette.
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Several independent clones were selected in G418 and a clone with low basal and a dose-dependent increase in luciferase activity upon infection with KSHV was selected for further analysis, and designated 293PAN-Luc.
In contrast, when tumors from an osteosarcoma clone with low expression of BMP-6 mRNA (CMT 353 B, clone 6; see Figure 1) were analysed, only weak, diffuse BMP-6 staining was observed (Figure 5D).
A clone with low (HsAQP8: 100:2) and one with high (HsAQP8: 1000:1) expression level were compared.
M21 cells express αvβ3 and αvβ5 (Wayner et al., 1991), and M21-L is a sub-clone with low αv expression (Cheresh and Spiro, 1987; Mitjans et al., 1995).
Comparison with a clone with low expression level showed that the majority of the protein ends up in the membrane fraction in both cases and, most importantly, that the membrane fraction from the highly expressing clone contains substantially larger amounts of recombinant protein.
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