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A total of 119 bands were obtained with molecular weights ranged from 2.77 to 131.88 kDa.
Bands were obtained with a LAS-3000 (FujiFilm, Tokyo, Japan) and quantified with NIH image J, version 1.37 analysis software (Scion image, NIH).
However, weak bands were obtained with two out of the 44 samples, most likely due to low DNA concentration.
Several additional bands were obtained with the C-terminal antibodies 2A10 and 4B11; however, these bands also appear in cells not overexpressing hMdm2 (not shown) and independent of the presence of LMB.
Electrophoresis on a 1% agarose gel clearly separated the PCR products into one to three bands: three bands were obtained with two primer sets (No. 10 and 12) (Type I), two bands with five primer sets (No. 3, 16, 18, 19 and 24) (Type II), and a single band with the remaining 17 primer sets (Type III).
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Optical density of the bands was obtained with Bio-Rad software (Quantity One; Hercules, CA).
Integrated density values for each band were obtained with an Alpha Innotech ChemiImager 5500 (Alpha Innotech, San Leandro, CA, USA), normalized with respect to the 28S band on ethidium bromide-stained rRNA loading patterns and expressed as the percentage change from control.
A single transcript band was obtained with HindII-linearized DNA, indicating that hilD is expressed from a single promoter (Figure 6A, lane 1).
Unexpectedly, a weak band was obtained with cDNA prepared from the thorax of C. solmsi.
The same band was obtained with the input DNA, whereas the normal IgG control and no antibody control did not result in the immunoprecipitation of DNA fragments detectable by PCR amplification.
The results were considered positive when there was at least an 8-fold difference in amplification versus the negative control (3-cycle difference in the Ct (cut-off of threshold) value) and a clear band was obtained with agarose gel electrophoresis.
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