Hello everyone!
I have searched everywhere for a simple protocol to isolate a nuclear protein fraction from yeast (Saccharomyces cerevisiae).
I want to do something very simple: look at the levels of my protein (via western blot) in the nucleus vs membrane associations in the cytoplasm. Currently, I have a differential centrifugation protocol that allows me to look at my protein of interest and whether it is in fractions associated with certain types of membrane. One of the first pellets I generate is full of nuclei and debris. Ideally, I would like to process that pellet to generate a nuclear protein fraction.
Many of the protocols I have found seem to be aimed at producing very pure extracts used for transcription assays, etc. These are very time-consuming and complicated (long dialysis times, etc.), and since I don't need this level of purity I would like a more basic protocol.
Does anyone have any suggestions? Or is my only option one of the more complicated protocols? I have attached an example of a nuclear preparation protocol that roughly represents the ones that I have found.
Thank you all for your help!
Hi,
Here's the method which my colleague has developed.
https://www.ncbi.nlm.nih.gov/pubmed/22465796
I hope it helps.
@Shin-Ichiro Hiraga
Thank you so much! I can't believe I haven't come across this publication...so helpful.
Just to confirm, I would stop at step (2) of 2.2.3. Chromatin preparation and directly load the supernatant to a SDS-PAGE gel? Or should I do a wash of EB buffer to get rid of the Triton X-100 and re-suspend in SDS Sample Buffer before loading?
2.2.3 is aimed to separate chromatin-associated proteins from soluble nucleoplasmic proteins.
Perhaps you can simply resuspend everything after 2.2.2 in SDS-PAGE sample buffer.
@Shin-Ichiro Hiraga
Yes, but wouldn't I have to still lyse the nuclei before proceeding?
That's why I was thinking I would continue until step #2 of 2.2.3 (which lyses the nuclei with Triton X-100 in EB buffer) but then just spin down and re-suspend in SDS sample buffer to proceed to SDS-PAGE.
Hi,
Processing the sample for SDS-PAGE (addition of SDS or LDS and boiling) will do that job.
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