Samples were diluted in 0.02M TEAB solution and sonicated with 40% intensity in 3 cycles of 10 s each using a Tip Sonicador Q125 (QSonica). Protein concentrates were quantified using Qubit™ method (Invitrogen). Aliquots containing 10 μg of protein were submitted to trypsin in-solution digestion, and the resulting peptide samples were desalted using homemade C18 reverse-phase microcolumns (22 (link)). The peptide concentration was also determined using Qubit™ protein assay.
Qubit method
Manufactured by Thermo Fisher Scientific
The Qubit™ method is a fluorescence-based technique used to quantify nucleic acids (DNA and RNA) and proteins in a sample. It provides accurate and sensitive measurements of biomolecule concentrations. The Qubit™ method utilizes fluorescent dyes that specifically bind to the target molecules, allowing for precise quantification.
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2 protocols using qubit method
1
Protein Extraction and Trypsin Digestion
2
Quantitative PCR for Ovarian CYP Expression
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An optimized end point PCR protocol was used to analyze the mRNA expression of CYP19A1 and CYP17A1. PCR was carried out in a final volume of 25 µL containing 10 ng cDNA (previously quantified by the Qubit method (Invitrogen)), 1.5 mM MgCl 2 , 0.5 µM forward primer, 0.5 µM reverse primer, 0.2 mM dNTP, Taq buffer 10× and 2 U Taq polymerase (5 U/ µL) (Invitrogen). The reaction conditions were as described in a previous study (Amweg et al. 2011) . Only granulosa samples positive for CYP19A1 mRNA and negative for CYP17A1 mRNA and theca samples positive for CYP17A1 mRNA and negative for CYP19A1 mRNA expression were used to detect CYP11B1 mRNA expression. In addition, the negative control performed using water instead of cDNA controls was negative to the reaction and the positive control using adrenal tissue was positive for CYP11B1 mRNA.
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