Then, the mixture remained at room temperature overnight with continuous stirring.
Membranes were then incubated with PKD1, PKD2, PKD3 antibodies (1∶1000 dilution) (PKD1 from Cell Signaling Technology, PKD2 & PKD3 from Bethyl Laboratories) overnight with continuous shaking at 4°C.
The dried plant material (~500 g) was ground prior to solvent extraction with a mixture of MeOH CH2Cl2 (1:1; v/v) overnight under continuous agitation at 20 °C.
Degradation products were removed by dialysis (Servapor 44146, Serva Feinbiochemica GmbH & Co., Heidelberg, Germany; cut-off 12 14 kDa) overnight under continuous movements against running deionized water <20°C.
The eluted fraction with the highest protein content was batch incubated with 200 μl immobilized capture mAb (200 μg MIF mAb per 100 μl of packed resin) overnight with continuous rocking at 4°C.
Following centrifugation at 14,000 g for 10 min at 4°C, 1 ml of clear lysates was incubated with 5 μl of rabbit anti-GSK-3β antibody or 5 μl of normal goat IgG overnight with continuous rotation at 4°C.
Final digestion was done using trypsin (Sequencing Grade, Promega, Madison, WI), at a ratio of 1 50 enzyme:substrate at 37°C overnight with continuous vortexing, followed by a second aliquot of trypsin, at a ratio of 1 100 enzyme:substrate, and an additional 2 hr of incubation.
Overnight there were continuous bursts of machine-gun fire.
Some patients require continuous overnight drip feeds or a bedtime dose of cornstarch in order to control their tendency to develop hypoglycemia.
Overnight use of continuous low-level heatwrap therapy for relief of low back pain.
After treatment with 1% BSA for blocking, the primary antibodies (Bcl-2, caspase-3, poly ADP ribose polymerase (PARP)) were incubated with the PVDF membrane at 4°C, overnight, followed by continuous incubation with secondary antibody for 1 h and stained with ECL.
