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The stable extracts were lyophilized in a speed-vac concentrator (Saur, Reutlingen, Germany) and stored at −20°C until analysis.
Peptides were identified by Orbitrap Velos mass spectrometry, from human sperm heat stable extracts and immunoprecipitates using rabbit anti-PPP1R2 antibodies.
PPP1R2P9 was originally found in cDNA libraries of human germ cell tumors, binding to PPP1C directly and in heat stable extracts inhibits this phosphatase potently with an IC50 of 0.2nM [ 40].
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Thermogravimetric analysis indicated that the most stable extract was basil, followed by rosemary and oregano.
For mass spectrometry analysis, the immunoprecipitate and the heat stable extract were resolved by 10% SDS-PAGE along with purified positive controls.
Also, an independent RIPA supernatant sperm extract was prepared, boiled in a water bath for 30 min, chilled on ice for 2 min and centrifuged at 16000 g for 20 min, 4°C to obtain a heat stable extract.
Recently, a report identified the PPP1R2 protein in heat-stable extracts of bull testis and mouse testis and sperm where it may account for this PPP1R2-like activity [ 58].
The wild-type protein was stable in extracts with no obvious degradation in 2 hr.
Usually, the IP address and location of the Web server is stable, so extracting the IP address and location of the Web servers by Web mining is an effective way to obtain landmarks [7], and because there are large numbers of Web servers in the network, a large number of landmarks can be obtained.
Field strain 34086b was serotyped by the immunodiffusion method using heat-stable antigen extracts [ 5] by Gallant Custom Laboratories (Cambridge, Ontario, Canada) as serotype 5.
We use this approach to analyse a set of robust and stable hairpins extracted from a genome-wide scan (Mendes et al., 2010) of Anopheles gambiae and Drosophila melanogaster, greatly reducing the number of candidates.
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