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actin in mitochondria

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Miri
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Topic Started by Miri
on 3/19/2009 15:36 PM   
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hi everybody,


i have a question a bit similar to one I've seen here: I do cell fractionation (animal tissue) and then western blot (the protocol is fine). there is a nice and clear band of b-actin and I wanted to use it for normalization (as I do for the cytosolic fraction), however someone told me that actin has "no business" being in the mitochondria....


HELP!


thank you all 


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varsha
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Posted By varsha
on 3/19/2009 19:58 PM   
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A small amount of actin with your mitochondrial fractin may be okay but you cant use it for normalisation.


If you are comparing mitochndria from different samples, you would have to use a resident mitochondrial protein for normalization.


To show purity of the fraction, use a mitochondria specific protein e.g cytochrome C. You may also have to show absence of  a soluble cytosolic protein e.g. GAPDH. Depends upon the question you want answered.


Good luck!



Miri
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Posted By Miri
on 3/19/2009 17:16 PM   
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Thank you,


I'm comparing mito from different samples (cytochrome c in the mitochondrial fraction), can't use any of the complexes for normalization since they differ in my treatments. Do you have any suggestions regarding possible normalisation protein


thanks again


"A small amount of actin with your mitochondrial fractin may be okay but you cant use it for normalisation.


If you are comparing mitochndria from different samples, you would have to use a resident mitochondrial protein for normalization.


To show purity of the fraction, use a mitochondria specific protein e.g cytochrome C. You may also have to show absence of  a soluble cytosolic protein e.g. GAPDH. Depends upon the question you want answered.


Good luck!"


 


Last edited Mar 19, 2009, 19:21 PM by R Bishop

R Bishop
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Posted By R Bishop
on 3/19/2009 17:28 PM   
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 Miri,


To clarify, are you comparing cytochrome c in two different samples by western blot to determine if the amount of mitochondria or complexes differs before and after treatment?


If so, Im not sure there is a proper loading control after spinning out the cell debris etc.  Im pretty sure you can just do, "pre-prep" beta-actin control to ensure you started with equal amounts of cells?  Otherwise I cant see what loading control there is, since by definition you're assay is attempting to show a total difference in mitochondria.


I'll do so digging anyway.


Rus


 

"Everything should be made as simple as possible, but no simpler."
-- Einstein



Miri
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Posted By Miri
on 3/19/2009 17:59 PM   
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Hi Rus,


I'm comparing cytochrome c in different mito samples to see whether there is an increase in this protein in my treatment and in a different gels I check cyto c in the cytosolic fraction because there is an apoptotic process involved.  I do load equal amount of protein (Bradford), however i'm used to normalize the cytosolic gels to actin antibody and thought to do the same with the mito. Can't start with cells cause work with tissue.


Miri


 [/quote]



heehawmcduff
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Posted By heehawmcduff
on 3/20/2009 9:56 AM   
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I've never done this and am just hypothesising but would it be possible to show normalised protein or loading usng a general stain like Ponceau?



R Bishop
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Posted By R Bishop
on 3/20/2009 7:35 AM   
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 Dang, you're so right heehaw thats exactly what you do.  Take an aliquot and silver stain a gel and quantitate the bands.


 


Rus

"Everything should be made as simple as possible, but no simpler."
-- Einstein



Miri
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Posted By Miri
on 3/20/2009 11:38 AM   
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Thank you all for your suggestions. I'll try.


Miri



varsha
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Posted By varsha
on 3/20/2009 17:14 PM   
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Hi Miri.


If you could get antibody to a mitochondrial membrane protein, that could be used for normalisation across samples from different tissues. I am thinking of components of mitochondrial translocase complex (TOM and TIM) which would not change much or leak out from the matrix. Any good affinity antibody might work. I would try labs which work on TOM and TIM. One of the names I recall is Ulrich F. Hartl.  Abcam has antibody to certain enzymes e.g aconitase for use as a marker. You could try it if you get them in trial size.


Varsha


 


Last edited Mar 23, 2009, 10:19 AM by varsha

Miri
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Posted By Miri
on 3/21/2009 6:41 AM   
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Hi Varsha,


I must say that I like your  antibody option better, and will definitely check with Abcam.


Thank you all very much


Miri  


[/quote]



varsha
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Posted By varsha
on 3/23/2009 11:20 AM   
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Great! Let us know if you find a good antibody that works in such assays.


Varsha



Miri
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Posted By Miri
on 3/24/2009 2:05 AM   
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 So here is an update:


 I've found few references for using a tubulin antibody as a control protein for mitochondrial fractions. Although I don't understand why tubulin can be used and actin can't (since they both are cytoskeleton proteins), I borrowed a tubulin antibody and it worked. So if somebody encounters a similar problem: tubulin is fine, however I still think that beta actin is O.K. as well (found an article from 1990 that reports beta actin purification from isolated mitochondria that was identical to the cytosolic beta actin.


Miri


: )



Flor
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Posted By Flor
on 1/4/2013 7:00 AM   
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Hi Miri, could you please  send me any of these references you have found?
"I've found few references for using a tubulinantibody as a control protein for mitochondrial fractions"

I used tubulin as loading control in mitochondrial fractions, and it worked, but I couldn ´t find bibliography that support my election.

Thanks!

Flor



Miri
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Posted By Miri
on 1/4/2013 9:15 AM   
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 Hi Flor, 
It's been a while, so I didn't find that tubulun article I wrote about, if I'll see it I'll let you know. We published with actin normalization, so if you need I could send you few ref that we used, here is one of them.
Good luck

Xilouri M, Papazafiri P. Anti-apoptotic effects of allopregnanolone on P19 neurons. Eur J NeuroSci. 2006;23(1):43–54. doi: 10.1111/j.1460-9568.2005.04548.x.



Flor
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Posted By Flor
on 1/7/2013 3:07 AM   
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 Thanks Miri!
    I found an article about the content of tubulin in mitochondria

Flor



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