500–2,000 chromosomes sorted into a 10 μl drop of PRINS buffer containing 2.5% (w/v) sucrose47on a microscope slide. Air-dried chromosomes were labeled by FISH with probes for the pSc119.2 repeat, Afa family repeat and 45S ribosomal DNA that allowed identification of all wheat andTh...
The annealed oligo was ligated into the BbsI-digested pX330 vector using 5 μl of 2× QuickLigation Buffer and 1 μl of QuickLigase (New England Biolabs). The ligation mixture was treated with Plasmid Safe exonuclease (Epicentre) and transformed in OneShot chemically competent Stbl3 cells...
M7911 5X Green GoTaq® Reaction Buffer 20ml 368 M7921 5X Colorless GoTaq® Reaction Buffer 20ml 368 M8221 LigaFast™ Rapid DNA Ligation System 30 reactions 249 M8225 LigaFast™ Rapid DNA Ligation System 150 reactions 899 M8291 GoTaq® Flexi DNA Polymerase 100u 173 ...
(B) FN-mediated tyrosine phosphorylation of Cas-L in Jurkat transfectants. Jurkat transfectants (A1, A2, Cas-L wild type; B1, B2, Cas-LΔSH3 mutant) were stimulated with PLL control or FN for 1 h and lysed on plates using 1% NP-40 lysis buffer. Lysates were immunoprecipitated with ...
The beads were washed four times in RIPA buffer, and equilibrated in kinase buffer (15 mM MOPS, pH 7.2, 7.5 mM glycerol 2-phosphate, 15 mM MgCl2, 3 mM EGTA, 0.15 mM dithiothreitol). The phosphorylation reaction was initiated by addition of substrate, histone H3 (1 μg) and ATP. In ...
Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AK
The resulting reaction was suspended in SDS-PAGE sample buffer (Bio-Rad, 1610747), boiled at 95 °C for 10 min, separated by SDS-PAGE, and then transferred onto a PVDF membrane for Western blotting. After blocking with 1% casein for 1 h, phosphorylated histones were detected by ...
Briefly, for each cell clone, three biological replicates of cultured cells (~5 × 106) were harvested, and cell pellets were lysed with about three volumes of lysis buffer (80 mM Tris-HCl, 4% SDS, 100 mM DTT, pH7.4). Cell lysates were sonicated to reduce the viscosity, and ...
The supernatant was centrifuged at 800 × g for 10 min; the pellet was washed by re-suspension in the same volume of SHEEP buffer yielding the crude nuclear pellet (P1) and pooled supernatants (S1) that were centrifuged at 9200 ×g for 10 min. The crude synaptosomal pellet was washed ...
The resulting immunoprecipitates were washed extensively in TNT and then kinase buffer (20 mm HEPES, pH 7.5, 20 mm MgCl2, 1 mm EDTA, 2 mm NaF, 2 mmβ-glycerophosphate, 1 mm dithiothreitol, 10 mm ATP). Precipitates were then incubated for 15 min at 30 °C in 20 μl of kinase ...