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Nuclear Extracts (Golgi contamination)


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#1 noakes84

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Posted 20 May 2009 - 03:12 AM

Hi there

I am trying to prove if my protein of interest is indeed localised in the nucleus. I have tried several methods (and commercial) kits to separate the nucleus intact from the cytosol. When I blot these fractions I do get nuclear specific proteins in the nuclear extract, so I know I can get that fine. The only problem is, is that the nuclear extract still must contain some Golgi membranes as blotting for Golgi-localised proteins (such as GM130) I get bands in both the cytoplasmic and nuclear fractions. I get bands for my protein of interest in both the cytoplasmic and nuclear fractions. This is a major headache as my protein of interest is also localised to the Golgi!

Does anyone know of a good way to seperate the Golgi from the nucleus? It seems that in all the papers I have read where they claim they have nuclear extracts, they never use a Golgi marker to show that it not there, or indeed care that Golgi membranes may still be present!

Help!!!

#2 lsek

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Posted 31 May 2009 - 04:05 AM

Hi,

It's nearly improbable to get a "clean" nuclear and Golgi fractions. They are rather "nuclear enriched" and "Golgi enriched" fractions. So, there will be carry overs. If your Golgi marker shows slight signal in the nuclear fraction, it's probably acceptable. You could determine if your target protein is localized in the nuclear or Golgi fraction by looking at the intensity of the signal.

After which, a cellular localization study, e.g. IFA (or others) would normally confirm or corroborate the subcellular fractionation result.

Edited by lsek, 31 May 2009 - 04:07 AM.

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#3 noakes84

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Posted 06 June 2009 - 12:06 PM

Hi,

It's nearly improbable to get a "clean" nuclear and Golgi fractions. They are rather "nuclear enriched" and "Golgi enriched" fractions. So, there will be carry overs. If your Golgi marker shows slight signal in the nuclear fraction, it's probably acceptable. You could determine if your target protein is localized in the nuclear or Golgi fraction by looking at the intensity of the signal.

After which, a cellular localization study, e.g. IFA (or others) would normally confirm or corroborate the subcellular fractionation result.


Thanks for that. What is this IFA you speak of? How does that work?

#4 noakes84

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Posted 06 June 2009 - 12:09 PM

Hi,

It's nearly improbable to get a "clean" nuclear and Golgi fractions. They are rather "nuclear enriched" and "Golgi enriched" fractions. So, there will be carry overs. If your Golgi marker shows slight signal in the nuclear fraction, it's probably acceptable. You could determine if your target protein is localized in the nuclear or Golgi fraction by looking at the intensity of the signal.

After which, a cellular localization study, e.g. IFA (or others) would normally confirm or corroborate the subcellular fractionation result.


Thanks for that. What is this IFA you speak of? How does that work?


Think im just being stupid there, thats just IF with a primary and fluorescent secondary Ab isnt it.....?




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