Cells were harvested 48 h later and analysed sequentially for firefly and Renilla luciferase activities by Dual-Luciferse Reporter Assay System (Promega Corporation,WIadison,WI).
The glass substrate of 1.1 mm in thickness is first washed by using liquid soap solution and sequentially cleaned for 10 min in an ultrasonic bath of acetone, ethanol, and deionized water, respectively.
Fifty micrograms of protein were separated by SDS-PAGE, blotted, and sequentially probed for total SMAD 2 3 (T-SMAD), phosphorylated SMAD 2 3 (P-SMAD), and GAPDH.
Potential CIRCLE participants were then randomly selected from each community's registry and sequentially contacted for consent until up to 50 people had agreed to participate.
This finding also confirms our previous observation that, in patients treated by HAART and sequentially tested for CRT, the co-receptor shift is independent of virological success [ 15].
Survey MS spectra were acquired for 1.5 s, and the three most intense ions (doubly or triply charged) were isolated and sequentially fragmented for 1.5 s by low-energy collision-induced dissociation.
For each method, the expected cases in a given month were defined by the previous 5 years of data and sequentially updated for each new observation year in the series.
Denatured E. coli soluble proteins were digested with trypsin (Promega) and sequentially enriched for phosphopeptides using TiO2 (Pierce TiO2 Phosphopeptide Enrichment and Clean-Up Kit) followed by IMAC (immobilized metal-affinity chromatography) (PHOS-Select Iron Affinity gel, Sigma Aldrich).
Immediately after hybridization the chips were placed in the Affymetrix GeneChip Fluidics Station 400 and sequentially processed for low stringency wash, followed by high stringency wash, streptavidin / phycoerythrin stain, repeat low stringency wash, anti-streptavidin antibody stain, a second streptavidin / phycoerythrin stain and a final low stringency wash.
