Overall, engineering arginines and phenylalanines for increased pKa and hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions.
Hybridization was at high stringency in Modified Church and Gilbert buffer overnight at 65°C.
Alignment of the probe to Xiap-ps1 showed 92% identity, indicating that the probe would be able to hybridise to both genes at high stringency.
All hybridizations were done at high stringency (i.e., hybridization at 65°C in 1M NaCl and final wash at 0.2×SSC at 65°C).
At high stringency the gamma HMM detected 39% of the chromosome 19 LTRs belonging to the aforementioned groups and detected by RepeatMasker (see Table 2).
Membranes were washed at high stringency, exposed on storage phosphor screens and visualized in a TYPHOON 9400 phosphorimage scanner (GE Healthcare, Bio-Sciences Corp).
These sites show strong conservation from Xenopus to man and were detected at high stringency by the Alibaba program (see Methods section) that predicts transcription factor binding sites.
The membrane was washed twice at high stringency (0.1× SSC, 0.1%SDS) at 60°C before analysis using a Typhoon Phosphorimager (Amersham Biosciences) and the ImageQuant software.
Any bands that hybridise at high stringency (65°C; 0.3x SSC) to the Xiap probe in the DNA from Xiap knockout mice indicate the presence of another gene similar to Xiap.
In fact, it was reported that even in XPD mutant HD2 cells, the dissociation of XPD and CAK from other TFIIH subunits was only detected when the immunoprecipitation was performed at high stringency [48].
At high stringency, only wild type TG2 and W241A-TG2 effectively bound to the GTP-agarose; which demonstrates that the W241A mutation has no effect on the guanine nucleotide binding ability of the protein (Fig. 2).
