TRIzol Reagent - Invitrogen

Dialyze the phenol ethanol supernatant (from the end of the DNA isolation protocol) against three changes of 0.1%
SDS at 4°C. Centrifuge the dialysate at 10000 g for 10 min. at 4°C and use the clear supernatant for Western blotting.
The phenol ethanol solution can dissolve some types of dialysis membranes (cellulose ester) so the dialysis tubing
being used should be tested with the membrane to assess compatibility.
Note on using SDS for dissolving the pellet: 1% SDS is required to resolubilize the proteins from the pellet; a lower
concentration will not be sufficient. After solubilization, the SDS may be diluted.
Additional Notes for Protein Isolation with TRIzol
Recovering phosphorylated proteins
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To recover Phosphorylated proteins, add a protease/phosphatase inhibitor to the 1% SDS before resuspending.
Membrane proteins
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Membrane bound proteins can also be extracted with TRIzol. Resolubilizing the protein is the difficult part depending on the
protein’s amino acid charge. A positively charged protein will go into solution easier than a lipophilic (hydrophobic) one. An
SDS or urea solution might not allow solubilization of the protein of interest.
Analysis of proteins before precipitation
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It is recommended to take aliquots of the ethanol/TRIzol phase prior to protein precipitation and run directly on SDS PAGE to
determine solubility/immuno reactivity of the proteins of interest. This eliminates the possibility of proteases being released when
the pellet is homogenized. Electrophoresis will separate out the phenol and ethanol from the protein. It is recommended that the
aliquot of the phenol/ethanol phase that is loaded must contain at least 10X the minimal amount of protein that is necessary for
detection of immunoreactivity: a suggestion is 2.5, 5, 10 µl of supernatant diluted in sample buffer. See Focus 17:3, 98; Wu, Lai-
Chu, Isolation and Long-Term Storage of Proteins From Tissues and Cells Using TRIZOL Reagent.
Interference of SDS with protein quantitation
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The SDS in the solubilization buffer can interfere with Bradford and Lowry Assays.Methods that do not have detergent-interface
problems, and that do not rely on A260/A280 measurements may be used (traces of phenol may cause overestimation of protein
concentrations).
Precipitation of SDS during dialysis
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If the SDS precipitates out during dialysis, remove the dialysis from the 4°C and place it at room temperature. This will allow the
SDS to go back into solution.
TRIzol LS Reagent
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