Samples were run and computer comparisons were generated by Kendrick Labs, Inc. (Madison, WI, USA), as described previously [30 (link)]. For this study, isoelectric focusing was carried out in a glass tube with an inner diameter of 3.3 mm using 2.0% pH 3–10 isodalt Servalytes (Serva, Heidelberg, Germany), and the gels were silver stained. Spot percentage is equal to a spot integrated density above a background expressed as a percentage of a total density above a background of all spots measured. Protein abundances were compared between developmental time points and between cultivars. Proteins identified as differentially expressed were required to meet the following specific criteria. The spot intensities were required to have a two-fold or greater change of the means with a p-value of less than 0.05 (n = 4). The data from both time points were included in the analysis when comparing the two cultivars and vice versa. For the comparison by developmental time point, a minimum two-fold change was required in both Sumner and Desirable samples individually (n = 2). Conversely, for the comparison by cultivar, a minimum two-fold change was required in both developing and mature time points individually (n = 2). These criteria were also met when using data from the Desirable 10/2 time point in place of the 9/25 time point. The sample in which the spot percentage was deemed the highest must have a spot percentage of at least one-fourth of the mean spot percentage of all the spots on the gel. Spots were visually confirmed for distinguishability and differential expression. Selected spots with significant differences in abundance were cut out and subjected to trypsin digestion to release peptides. Proteins within selected spots were identified by mass spectrometry, as described above.
Pecan Nut Protein Profiling
Samples were run and computer comparisons were generated by Kendrick Labs, Inc. (Madison, WI, USA), as described previously [30 (link)]. For this study, isoelectric focusing was carried out in a glass tube with an inner diameter of 3.3 mm using 2.0% pH 3–10 isodalt Servalytes (Serva, Heidelberg, Germany), and the gels were silver stained. Spot percentage is equal to a spot integrated density above a background expressed as a percentage of a total density above a background of all spots measured. Protein abundances were compared between developmental time points and between cultivars. Proteins identified as differentially expressed were required to meet the following specific criteria. The spot intensities were required to have a two-fold or greater change of the means with a p-value of less than 0.05 (n = 4). The data from both time points were included in the analysis when comparing the two cultivars and vice versa. For the comparison by developmental time point, a minimum two-fold change was required in both Sumner and Desirable samples individually (n = 2). Conversely, for the comparison by cultivar, a minimum two-fold change was required in both developing and mature time points individually (n = 2). These criteria were also met when using data from the Desirable 10/2 time point in place of the 9/25 time point. The sample in which the spot percentage was deemed the highest must have a spot percentage of at least one-fourth of the mean spot percentage of all the spots on the gel. Spots were visually confirmed for distinguishability and differential expression. Selected spots with significant differences in abundance were cut out and subjected to trypsin digestion to release peptides. Proteins within selected spots were identified by mass spectrometry, as described above.
Corresponding Organization : Southern Regional Research Center
Other organizations : Oak Ridge Associated Universities, James Madison University, Noble Research Institute, New Mexico State University
Variable analysis
- Time points (9/18, 9/25, 10/2)
- Protein abundance
- Spot percentage
- Cultivars (Sumner, Desirable)
- Biological replicates (2 per time point per cultivar)
- Protein extraction from 3 individual nuts per biological replicate
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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