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Estimates suggest that at distances of 100 to 200 miles (160 to 320 km) inland from the barrier, 15 to 20 inches (380 to 500 mm) of ice may be added to the shelf each year by bottom freezing.
An extensive literature survey is made, and utilizing a finite difference algorithm suggested by Du-Fort Frankle, the cylindrical coordinate system based numerical model is developed and the time duration for solidification of 10 mm of ice around a 20 mm diameter pipe is found to be 2609.4 s.
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Next, 5 × 10−2 mM of ice-cold NaBH4 solution was added to the solution while stirring.
The reaction was stopped by the addition of EDTA to a final concentration of 10 mM on ice, and the sample was centrifuged at 10,000 g for 10 min at 4°C to yield an S1 supernatant and a pellet.
When required, soluble GTPases were loaded with nucleotide in solution by addition of GDP or GTPγS (20× molar excess) and EDTA (5 mM final concentration), followed by incubation at 30°C for 30 min and addition of MgCl2 to a final concentration of 10 mM on ice.
The digestion reaction was stopped by the addition of EDTA to a final EDTA concentration of 10 mM (on ice).
After 20 min at 37°C, MgCl2 was added to a final concentration of 10 mM on ice.
Immunoprecipitates were washed in 0.1% NP-40, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na3VO4 supplemented with protease inhibitor and a final wash of ice cold, 10 mM Tris-HCl, and pH 7.5.
Beads were washed twice with 200 µl of ice cold 20 mM MOPS, 4.5 mM MgAcetate, 150 mM KCl, 0.2% Tx100.
Briefly, 10 volumes of ice cold 5 mM Tris/0.1 mM Na2EDTA, pH 7.6 were added to each of the tubes containing buffy coat free – packed erythrocytes of diabetics and non-diabetic samples to achieve osmotic lysis.
Neutrophils were then resuspended in 30 µl of ice cold buffer (50 mM Tris, 50 mM NaF, 50 mM β-glycerophosphate, 10 mM Sodium Orthovanadate) with 1∶100 protease inhibitor cocktail set IV and 1 mM PMSF.
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