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Modified Lowry Miller Protein Assay Wash Step for Nuclei
 

Summary: 93
Modified Lowry Miller Protein Assay
Wash Step for Nuclei
For optimum accuracy with nuclei, the Ficoll must be removed.
1. Pipette 50 l of nuclei into 500 l 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 4C.
4. Carefully remove all supernatant.
5. Resuspend pellet in 50 l 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 l. For yeast protein lysates in SDS-
PAGE sample buffer, try 2 l of sample + 98 l ddH2O.
2. Add 50 l 0.05% NaDeoxycholate. Vortex well.
3. Incubate at room temperature for 5 minutes.
4. Add 150 l 10 % TCA. Vortex well.
5. Spin in microfuge 5 minutes at top speed at room temperature.
6. Carefully remove all supernatant.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine