Lue staining and Western blot evaluation making use of antibody specific for the 6 His tag. The protein concentration was measured by the BCA technique and stored at 70 until further use. Single-stranded exchange assay. The assay mixture contained reaction buffer (25 mM Tris-HCl, pH 7.five, and five glycerol), ten mM MgCl2, 5 M circular X 174 RF I dsDNA (New England Biolabs [NEB]), 15 M X virion dsDNA (NEB), 1 mM DTT, and 2 M concentrations with the proteins (RecA obtained from NEB M0249S, PfRad51, or PfRad54) to be tested for functional activity. The reaction mixture (135 l) was preincubated at 37 for ten min, followed by addition of 15 l of an initiation mixture (final concentration of 25 mM Tri-HCl, pH 7.five, five glycerol, three mM ATP, and 0.five M single-stranded binding protein [SSB from Stratagene or PfRPA1L or PfRPA1S]). Aliquots have been collected at indicated time points and quenched with quit resolution (1 SDS, 15 mM EDTA), along with the merchandise were analyzed by 1 Tris-acetate-EDTA (TAE) agarosembio.asm.orgMay/June 2013 Volume four Concern 3 e00252-Recombination and DNA Harm Repair in Parasite Growthgel electrophoresis.Ifosfamide The gel was run overnight at 18V, stained for 1 h with ethidium bromide (final concentration, 0.5 g/ml), and visualized soon after destaining in water. Parasite culture, RNA extraction, and cDNA synthesis. P. falciparum clone 3D7 was grown at 4 hematocrit in RPMI 1640 medium supplemented with 10 O standard human serum and red blood cells.Tirofiban Parasites had been cultured at 37 making use of the candle jar strategy (42) and visualized by Giemsa stain (43).PMID:23554582 Parasite cultures were synchronized by the sorbitol approach as described previously (44). Synchronized ring-, trophozoite-, and schizont-stage parasites have been treated with 0.005 and 0.05 methyl methane sulfonate (MMS) inside the culture medium at 37 , and aliquots had been collected for RT-PCR and Western blot evaluation. RNA was extracted from MMS-treated and untreated parasites employing Trizol reagent (Invitrogen), and the concentration of RNA was measured making use of NanoDrop (model 2000/2000c). One particular microgram of RNA was reverse transcribed employing oligo(dT)18 primer, a first-strand cDNA synthesis kit (Roche Diagnostics GmbH), and cycler protocol conditions as described by the manufacturer. The resulting cDNAs have been subjected to PCR amplification for 30 cycles using gene-specific primers. Handle reactions with out reverse transcriptase were carried out to confirm that no DNA contamination was present within the RNA samples. The merchandise have been run on 1 agarose gel and stained with ethidium bromide. Western blot evaluation. MMS-treated synchronized parasites were released from red cells by 0.01 saponin lysis for five min at 0 and washed two times in ice-cold phosphate-buffered saline (PBS). The samples have been analyzed by Western blotting making use of a 1:five,000 dilution of mouse antiPfRad51 (M. Bhattacharyya and N. Kumar, unpublished data), a 1:five,000 dilution of mouse anti-ScRad54 (a type gift from Wolf-Dietrich Heyer, UC, Davis), and a 1:7,500 dilution of mouse anti-PfHsp70 (45) antibody applying enhanced chemiluminescence (ECL)-based detection solutions (Amersham Pharmacia Biotech). Comet assays and DNA damage repair. MMS-treated synchronized parasites corresponding to four 105 infected red blood cells have been collected, lysed with 0.01 saponin, washed 3 times with cold PBS, and finally resuspended in 1.0 ml PBS for single-cell gel electrophoresis (Comet assays) (46). Diluted cells (30 l) have been mixed with 300 l Comet LMAgarose (1 low-temperature melting agarose [Tr.