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Reaction was stopped by adding equal volume of SDS-Urea-EDTA buffer (2× SDS loading buffer with 8 mol/L urea and 15 mmol/L EDTA) and incubating at 90°C for 5 min. Samples were cooled to room temperature and 0.5 μg of proteinase K was added for a 5 min incubation at 37°C.
The reactions were terminated after 15 min by adding equal volume of 100% formamide containing traces of bromophenol blue.
Sample was diluted by adding equal volume of B88 and centrifuged again at 34,000 rpm for 1 hr at 4°C.
For 2-DE analysis, apoplast proteins were precipitated from the extract by adding equal volume of 20% TCA in acetone containing 0.5% DTT as described by Haslam et al. [69] and incubated overnight at −20°C.
Biotinylated gEt protein was incubated with rIDE or control proteins at room temperature or 37°C for 30 min followed by adding equal volume of 6 M urea for 18 hr at room temperature.
Subsequently the media was removed and cells were lysed by adding equal volume of dimethyl sulfoxide.
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Following centrifugation, 400 µl of the upper aqueous phase was transferred into a clean microfuge tube and RNA was precipitated by adding equal volumes of ice cold isopropanol.
Simultaneous overexpression of EndoG-FLAG and Bcl-xL was achieved by adding equal volumes of viral stocks for each gene at 6 hours in vitro, washing and replacing with the required medium 24 hours later.
The siRNA complex was formed in the 48 well plate by adding equal volumes of the stock concentration of siRNA and OptiMEM and incubating at room temperature in a laminar flow hood.
For LPS stimulation, the splenocytes from 1-month old MRL-lpr mice were adjusted to 5×106 cells/ml, plated in 24-well plate, and then activated for 24 hrs by adding equal volumes of media containing LPS (Sigma, 1000 ng/ml) to seeded cells (final concentration at 500 ng/ml).
Finally, AP activity was determined by adding equal volumes of AP-substrate buffer (2 M diethanolamine pH 9.8, 1 mM MgCl2, 6.7 mg/ml Sigma 104 phosphatase substrate) to 20 µl cell lysates in 96-well microtiter plates and measuring the absorbance at 405 nm in an ELISA reader (Multiskan RC, Labsystems).
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