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The M-S-PB mixture contains PS beads at a volume fraction of 2.0%%.
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In this report, the beads were injected at a volume of 4.6 or 23 nL by serial injection of the unit volumes (2.3 nL or 4.6 nL).
Abs to Shc protein (3 µg) and to ubiquitin (20 µl) were first respectively incubating with Protein A-Sepharose beads (50 µl of 10% suspension) in 500 µl of lysis buffer for 1 h at 4°C. Cell lysates (0.3 mg) were incubated with Abs coated in Protein A-Sepharose beads in a volume of 500 µl at 4°C for 2 h.
200 µl lysate was incubated with GST-VAMPs, GST alone, syntaxin16-PrA or PrA alone bound to the appropriate beads in a volume of 1 ml at 4°C with rotation.
Purified DDX3X helicase domain (3 µM) was added to the beads in a volume of 1 mL and incubated for 3 h at 20°C with gentle rotation.
A suspension of streptavidin-agarose beads in a volume of 80 μl was added.
The chain length is fixed at 12 beads at every volume fraction (including 1/1, 1/4, 1/6, 1/14, and 1/20).
We pooled these reactions and did two rounds of PCR cleanup using Agencourt AMPure beads at a 0.8 bead-mixture:sample-volume ratio.
Arg was immobilized on Affigel 10 beads (Bio-Rad) according to the manufacturer's directions, and GST-tagged Arg fragments and mutants were bound to glutathione agarose beads at a concentration of 20 μM (moles of protein per volume of beads).
The two bead batches were further diluted with PBS to a final concentration of 400 μg/mL, after which they were mixed at a volume to volume ratio of 1 2 to get final approximate concentrations of 130 and 270 μg/mL of 250- and 100-nm beads, respectively.
Based on these volume estimations, an injection of 4.6 nL of beads was equivalent to a volume of 5.3% or 1.4% of the total volume of the ventricles at stages 46 and 50, respectively.
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