Axonemal pellets isolated from heavily ciliated cultures as described above were resuspended in SDS sample buffer and loaded onto 4–12% Nu-PAGE gradient gels (Novex) for separation. Axonemes from three different cell donors were analyzed independently. Following staining with Coomassie Blue, the entire lane from each replicate was cut into 18 sections, the width of the section dependent upon the intensity of protein bands present in that region of the gel, with the goal of equalizing to the extent possible the amount of protein contained in each band (Fig. SI-2 ). In most cases, multiple visible bands were contained within a given section, with the exception being the thick, intense band corresponding to highly abundant α- and β–tubulin at approximately 50 kDa. Each section was subjected to in-gel trypsinization using the protocol of Wilm et al. (50 (link)), and a predigested BSA standard (MassPrep Standard, Waters Corporation) was spiked into each digested band to provide a level of 250 fmol BSA per injection.
Massprep standard
Manufactured by Waters Corporation
The MassPrep Standard is a lab equipment product manufactured by Waters Corporation. It is designed to provide accurate and consistent sample preparation for mass spectrometry analysis. The core function of the MassPrep Standard is to automate the sample preparation process, ensuring reproducible results.
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Lab products found in correlation
2 protocols using massprep standard
1
Axonemal Protein Extraction and Proteomic Analysis
2
Hybrid Proteome Samples for MS Analysis
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Commercial tryptic digests of S.cerevisiae (Yeast, Promega, #V746A), human K562 cells (Promega, #V695A) and E.coli (MassPREP standard, Waters, #186003196) were reconstituted in 2% ACN/0.1% FA to final concentration of 0.1 μg/ul. To generate the hybrid proteome samples, purified peptides from each of the three species were combined in different proportions as previously described [42 (link)] and as follows: sample YHE211 consisted of 30% w/w Yeast (3 μg), 65% w/w Human (6.5 μg) and 5% w/w E.coli (0.5 μg); sample YHE114 consisted of 15% w/w Yeast (1.5 μg), 65% w/w Human (6.5 μg) and 20% w/w E.coli (2 μg); sample YHE010 consisted of 0% w/w Yeast (0 μg), 100% w/w Human (6.5 μg), and 0% w/w E.coli (0 μg). Ten replicates of each proteome mixture were subjected to LC-MS/MS analysis on a timsTOF Pro mass spectrometer.
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