The Most Important Fulvestrant Capture Method

The proteins were detected through RRAD silver staining. Protein Visualization and Image Analysis The stained gels were scanned and digitized using a Duoscan scanner (Agfa, Trenton, NJ, USA; Bio-Rad, Hercules, CA, USA). After background subtraction, normalization, and matching, the spot volumes in gels from each treated-cell sample were compared with the matched spot volumes in gels from control cells. Comparison of the test spot volumes with the corresponding standard spot volumes yielded a standardized abundance for each matched spot, and the values were averaged across triplicates for each experimental condition. Statistical analysis was performed to select the matching spots across all images, including spots displaying a �� 1.5 average-fold increases in abundance between conditions and spots with P min. After removing all liquid, acetonitrile was added to cover the gel pieces. Acetonitrile was removed after the gel pieces were shrunk. The gel pieces were rehydrated in 0.1 M ammonium bicarbonate for 5 min, and subsequently incubated for 15 min with an equal volume of acetonitrile. After removing all liquid, the gel pieces were dried in a vacuum centrifuge for 20 min. The gel pieces were swollen in 10 mM DTT/0.1 M ammonium bicarbonate and incubated for 45 min at 56��C, followed by cooling at RT. After removing the excess liquid, the same volume of freshly prepared 55 mM iodoacetamide in 0.1 M ammonium bicarbonate was added, followed by incubation in the dark for 30 min at room temperature. The iodoacetamide solution was removed, and the gel pieces were incubated in 30 mL of 0.1 M ammonium bicarbonate for 5 min, and subsequently further incubated for 15 min with an equal volume of acetonitrile. After an additional incubation with ammonium carbonate/acetonitrile, the gel pieces were dried in a vacuum centrifuge for 20 min, rehydrated in digestion buffer and placed on ice for 45 min. The buffer was replaced with 20 mL of digestion buffer with trypsin (12, 500 ��g mL-1). After overnight digestion at 37��C, a sufficient volume of 25 mM ammonium bicarbonate was added to cover the gel pieces and incubated for 15 min. The same volume of acetonitrile was added and incubated for 15 min, followed by the addition of 5% formic acid/acetonitrile (1:1) to the recovered supernatant and incubation for 30 min. After repeating this step, all the extracts were dried in a vacuum centrifuge for 1�C2 h.