Labeled samples (250 µL) were applied to IPG strips (13 cm, pI ranges 3–10, GE-Healthcare) on the rehydration tray (GE-Healthcare) and focused using an Ettan IPGphor II (GE-Healthcare) as follows: active rehydration at 30 V for 14 h, followed by isoelectric focusing for a total of 28 kV/h (step to 500 V for 1 h, step to 1000 V for 1 h, step to 8000 V to a total of 28 kV/h). After isoelectric focusing, disulfide bonds were reduced by placing the strips for 10 min in 20 mL equilibration buffer (6 M urea, 50 mM Tris, pH 8.8, 30% glycerol, 2% SDS) containing 5 mg / mL DTT. The strips were then incubated for 10 min in fresh equilibration buffer with 45 mg / mL iodoacetamide. For the second dimension, the IPG strips were placed on 12% homogeneous polyacrylamide gels (4% stacking). Gels were cast using low-fluorescence glass plates (13 cm plates, GE-Healthcare) previously treated with bindsilane (GE-Healthcare). Each SDS-PAGE was run at 9 mA for 16 h in a HOEFER SE-600 system. Individual images of Cy2-, Cy3- and Cy5-labeled proteins of each gel were obtained using a Typhoon 9410 scanner (GE-Healthcare) with excitation / emission wavelengths of 480 / 530 nm for Cy2, 520 / 590 nm for Cy3 and 620 / 680 nm for Cy5. After imaging the gels were stained with colloidal Coomassie (Bio-Rad, Hercules, CA, USA).
Rehydration tray
Manufactured by GE Healthcare
The Rehydration tray is a piece of laboratory equipment used to maintain the hydration of samples or specimens. Its core function is to provide a controlled environment to keep samples moist and prevent them from drying out during storage or transportation.
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2 protocols using rehydration tray
1
Two-Dimensional Gel Electrophoresis Protocol
2
Saccharomyces cerevisiae Protein Extraction
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The Saccharomyces cerevisiae BY4741 strain was grown in YPD medium till 0.7 OD600 then 0.02% MMS treatment was done for 6 h. The cells were collected by centrifugation at 5000 rpm for 5 min and washed with phosphate buffered saline (PBS, pH7.4). Approximately 500 mg cells were suspended in 2.0 ml of protein extraction buffer (20 mM Tris, 4% CHAPS, 2.5% glycerol and 1X PIC). Cells were lysed using French press (30 Kpsi) and the supernatant was collected by centrifugation at 14,000 rpm for 30 min at 4 °C. The proteins in the supernatant were precipitated with 10% (v/V) TCA and incubated overnight at −20 °C. The precipitated protein was collected after centrifugation at 14,000 rpm for 15 min. The pellets were washed thrice with ice chilled acetone. Finally, the protein pellet was suspended in 300 μl of rehydration buffer (7 M urea, 2 M thio-urea, CHAPS 4%, DTT 10 mM, Tris 20 mM). Pellets were briefly sonicated in rehydration buffer and incubated for 2 h. Protein concentration was estimated and samples were stored at −80 °C. Approximately, 250 μl rehydration solution containing 250 μg protein was applied in rehydration tray and IPG strip (nonlinear, 11 cm, 3–10 pH range) was placed on rehydration solution mixture for strip rehydration. Samples were rehydrated passively for overnight in rehydration tray (GE healthcare).
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