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Is there alpha-tubulin in the nucleus?


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#1 nocas

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Posted 27 August 2010 - 08:28 AM

Hi!

I recently used alpha-tubulin to see if there was cytoplasmic contamination in my nuclear extracts and the corresponding band appeared. So there are two hypotheses: either my extracts are contaminated (can anyone suggest me a protocol to prepare nuclear extracts that guarantees no cytoplasmic contamination?) or alpha-tubulin is present in the nucleus. I'm hoping someone knows the answer. By the way, I use a monocytic cell line ( THP-1).
Thanks in advance

#2 Curtis

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Posted 28 August 2010 - 03:05 AM

what is your method of isolation? I think you have contamination in your fractions.

Edited by Curtis, 28 August 2010 - 03:05 AM.


#3 bob1

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Posted 29 August 2010 - 01:44 PM

The best bet for "pure" nuclei is to use a homogenizer protocol, followed by a sucrose gradient. Many of the cell swelling followed by sucrose gradient protocols don't seem to work very well.

#4 nocas

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Posted 30 August 2010 - 02:59 AM

I found an extraction protocol suggested by Sigma. It is not very different from the one I use by I'll try. I send it in attachment in case you want to take a look and give your opinions.
Back to alpha-tubulin, I would like to test it in a commercial nuclear extract sample to know, for sure, if this protein exists in nucleus. Do you know any company that sells this kind of product?

Thanks
Attached File  Nuclear extraction.pdf   68.46KB   1402 downloads

#5 laurequillo

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Posted 30 August 2010 - 04:31 AM

Hi!

I recently used alpha-tubulin to see if there was cytoplasmic contamination in my nuclear extracts and the corresponding band appeared. So there are two hypotheses: either my extracts are contaminated (can anyone suggest me a protocol to prepare nuclear extracts that guarantees no cytoplasmic contamination?) or alpha-tubulin is present in the nucleus. I'm hoping someone knows the answer. By the way, I use a monocytic cell line ( THP-1).
Thanks in advance


We use that antibody and it should not appear in the nuclear fraction. One thing you should do in to wash extensively between the citosolic extraction and the nuclear extraction. So you precipitate the nuclei and you collect the citosolic fraction right? and then you wash the nuclei before the nuclear extraction; you can wash more times to be sure you are getting rid of the citosolic fraction.
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