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Summary: 93
Modified Lowry Miller Protein Assay
Wash Step for Nuclei
· For optimum accuracy with nuclei, the Ficoll must be removed.
1. Pipette 50 µl of nuclei into 500 µl of 1XPM buffer (no PICs). Repipette to wash nuclei into buffer.
2. Vortex well.
3. Spin in microfuge at top speed for 30 minutes at 4°C.
4. Carefully remove all supernatant.
5. Resuspend pellet in 50 µl ddH2O. Vortex and bath sonicate briefly. Keep on ice until assay.
TCA Precipitation
· Do the TCA precipitation for protein samples with interfering substances, such as detergents, DTT,
ammonium sulfate, etc. If unknown samples are precipitated with TCA, then BSA aliquots for the
standard curve must be TCA precipitated likewise.
1. Add sample to 1.5 ml microfuge tube, and add ddH2O to 100 µl. For yeast protein lysates in SDS-
PAGE sample buffer, try 2 µl of sample + 98 µl ddH2O.
2. Add 50 µl 0.05% NaDeoxycholate. Vortex well.
3. Incubate at room temperature for 5 minutes.
4. Add 150 µl 10 % TCA. Vortex well.
5. Spin in microfuge 5 minutes at top speed at room temperature.
6. Carefully remove all supernatant.
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