How about super resolution confocal microscopy?
Sent from my iPhone, Fred Spiegel
> On Jun 5, 2021, at 4:00 PM, Rob Kay <[log in to unmask]> wrote:
>
> For what it's worth, and based on my experience with rat liver homogenates
> long ago, I would not even try fractionation!
>
> It's worth doing a very basic fractionation into nuclear/mitochondrial,
> endoplasmic reticulum and high speed sup fractions, but beyond that, I
> think it is very hard to get definitive assignment to Golgi, contractile
> vacuole, or vehicles of the endocytic pathway. The various sorts of
> gradient you can use will give broad, overlapping fractions and if you are
> looking at vesicles, you will have to work hard to keep them intact.
>
> Better to improve your tagging or antibody......
>
> Rob
>
>
>> Dear Dicty-colleagues,
>>
>> We are trying to figure out the subcellular localization of a membrane
>> protein. Due to tagging and expression issues, we are not able to do this
>> by microscopy. However, we can detect the protein by Western blot. So we
>> are wondering if it is possible to determine the localization by
>> subcellular fractionation.
>>
>> Does anyone have a good protocol for separating different organelles in
>> Dicty? Such as sucrose or percoll gradient?
>>
>> Many thanks in advance,
>>
>> Huaqing
>>
>> _________________________________________
>>
>> Prof. Dr. Huaqing Cai
>> Institute of Biophysics
>> Chinese Academy of Sciences
>> 15 Datun Rd, 6223, Chaoyang District
>> Beijing
>> China
>>
>>
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