I am having problems with gradient formation. I am using 6 mL of Percoll 80%, over it 6mL Percoll 35% and then in the top the buffer resuspension approx 35 mL (leaves extract in NIB) -> all in a 50mL Falcon tube. After centrifugation during 1h, at 2,000xg, 4C, using swing bucket and both the longest acceleration and desaceleration; I am not observating the gradient phases clearly. Any advices?
Nulcei Isolation buffer (NIB): 10mM NaCl, 10mM KCl, 2.5mM EDTA, 250 mM sucrose, 0.1mM spermine, 0.5 mM spermidine and 1 mM DTT.
Triton was added to 1%.
these references may help:
https://www.researchgate.net/post/How_to_avoid_chloroplast_contamination_during_nuclei_isolation
see also: http://www.plantmethods.com/content/pdf/1746-4811-9-31.pdf
We performed a nuclear subproteome analysis using the following for obtaining a "nuclear protein enriched fraction":
Cells were collected and washed with phosphate buffered saline (PBS). The cells were suspended in 1 mL of the isotonic buffer containing 10 mM HEPES-NaOH, pH 7.5, 0.25 M sucrose, 1 mM EGTA, and protease inhibitors. For cell lysis via plasma membrane solubilization, Triton X-100 at final concentration of 0.25% was added. Samples were vigorously stirred for 5 s and incubated 15 min at 4 °C. The cell lysate was centrifuged for 15 min at 12,000 × g at 4 °C. Nuclear pellets were washed with 500 µL of a solution containing 10 mM HEPES-NaOH, pH 7.5, 0.34 M sucrose, 0.75 mM spermidine, 0.15 mM spermine, 1 mM EDTA, 0.1% Triton X-100, and protease inhibitors, centrifuged at 1000 × g for 5 min at 4 °C, and decanted. The nuclear proteins enriched pellets were then solubilized in Tris/HCl 50 mM, pH 8.0, SDS 1%, DTT 2% and incubated 1 h at 37°C.
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