I want to isolate a good quality of protein from neuronal cell lines. I want to know about the effective lysis buffer that does not give any artifacts.
Do you want to generate subcellular fractions, ie Mito, Cyto and Nuclear- or you just need to ensure that the nuclear proteins are liberated? If you just need to ensure that the nucleus ruptures then RIPA buffer is the easiest selection. You can experiment with volumes, but a quick rise with PBS and then 300ul per well/6well (or equivalent) and scrape with a rubber policeman. If you want fractions, then I would suggest starting simple with the protocol by abcam - which we have used in our lab. Subcellular fractionation methods can be very complex and better to prove the simple version doesn't work before moving up. Artifacts can be the product of many steps during western blotting, but we use RIPA with many types of cell lines and find good results.
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