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The Nucleospin kit (Bioké, Leiden, The Netherlands) was used to clean the cDNA samples to reduce unspecific binding to the arrays.
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The kinetics of antigen binding to the arrayed antibodies can be used to determine the concentration of a specific protein (for example the tumor marker β2-microglobulin) in solution for a broad range of analyte concentrations.
Nevertheless, the collective patterns of autoantibodies binding to the arrayed self-antigens were informative.
Analysis of our initial BAC hybridizations revealed that a high proportion of transposon-like sequences and long and short interspersed nuclear elements were binding to the array.
Although only short fragments were hybridized, secondary structures formed either within one sample molecule or between different targets could cause inefficient binding to the array-bound probe molecules.
These results show that despite some differences between binding to the arrayed and fusion proteins, NHERF2 is targeted by Map, EspI and NleH1.
Protein concentration in the printing buffer and protein binding to the array demonstrated a characteristic saturation curve, with protein concentrations of over 1 μg/mL achieving >90% maximum signal intensity (Fig. 1E).
H-NS was found to be a key protein for these processes; presumably inactivating triplet repeats transcription by differently binding to the repeat arrays and then promoting IS1E transposition (Figure 6).
Thus, the lack of binding to the glycan array reflects an alternative structural mechanism of optimizing interactions with sugars, in this case with minimal local selectivity so that spatial distribution becomes the dominant factor in determining specificity.
However, closer gene by gene inspection showed that one Affymetrix feature, Gga.17679.1.S1_s_at, was mapped to 17 Ensembl Gene IDs from the 30 un-annotated genes above, indicating again high sequence homology between these genes that allowed binding to the same array probe.
We monitored a chimeric protein containing the ETS domain of GABPα fused to GST (GABPα-GST) binding to the ETS-CRE array using a fluorescent antibody against GST (Badis et al. 2009).
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