We used TRIzol to extract RNA and proteins from some tissue (that we no longer have any left of), and in trying to run some westerns found that TRIzol is terrible for westerns. Our western protocol works fine on (somebody else's) RIPA extracted proteins. We know we have proteins- looks good in the Pierce protein assay
Does anybody have a rescue for TRIzol extracted proteins so that we can run them in a Western? Specifically interested in hearing from someone who has done this. Thanks so much
What doesn`t work exactly? No protein on the membrane?
Am I assuming right that you have the Protein precipitated from the Phenol/Ethanol Supernatant of the TRIzol with Isopropanol and washed it afterwards? And you are now trying to do a SDS-PAGE + Western?
Have you added a suitable buffer to the proteins that contains SDS?
We have no protein on the membrane or on the gel itself (imaged using BioRad Criterion stain free tech). We ran a positive protein control with our samples and we get good transfer with those, so it is not a problem with the western protocol, but rather with our protein extraction. We followed the Trizol extractions with EtOH/guanidine washes, and then solubilized with an SDS buffer.
This is very odd... did you have a visible precipitate after the EtOH/guanidine precipitation/wash?
Maybe this can help to find a solution?Article A Unique Method for Isolation and Solubilization of Proteins...
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