Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Cell Biology and Cell Culture
Register Search Today's Posts Mark Forums Read

Cell Biology and Cell Culture Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


Intact Mitochondria isolation

Intact Mitochondria isolation - Cell Biology and Cell Culture

Intact Mitochondria isolation - Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 08-29-2011, 03:10 PM
Pipette Filler
Points: 373, Level: 7 Points: 373, Level: 7 Points: 373, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Location: Grenoble france
Posts: 17
Thanks: 3
Thanked 4 Times in 4 Posts
Default Intact Mitochondria isolation



I'm looking for a good protocol to purify intact mitochondria from cultured cells.
I have used a lot of different kit, but the dounce homgenization part is to crtical, and I obtained a lot of broken mitochondria and the sample was not really pure.
I'll planed to use the pierce kit

Have you some good advices for me.


thanks
Reply With Quote
  #2  
Old 08-31-2011, 06:00 PM
Warthaug's Avatar
Post-Doc
Points: 2,383, Level: 31 Points: 2,383, Level: 31 Points: 2,383, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Location: London, Canada
Posts: 203
Thanks: 1
Thanked 68 Times in 61 Posts
Default Re: Intact Mitochondria isolation

I've used the kits - they suck; save your money.

Isolating mitochondria is not trivial, and I need to know more before I recommend a protocol. Please answer the following questions:
1) What cell type are you isolating from?
2) How many cells (or what sized plate) are you preforming the isolation from?
3) How pure do you need them to be?
4) What is the intended use (i.e. what quantities do you need, any how healthy do they need to remain.

Bryan
Reply With Quote
The Following User Says Thank You to Warthaug For This Useful Post:
Nemesis05 (09-01-2011)
  #3  
Old 09-01-2011, 06:51 AM
Pipette Filler
Points: 373, Level: 7 Points: 373, Level: 7 Points: 373, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Location: Grenoble france
Posts: 17
Thanks: 3
Thanked 4 Times in 4 Posts
Default Re: Intact Mitochondria isolation

Thanks for you answer

1) What cell type are you isolating from?
Hela cells
Cos 1
CHO
2) How many cells (or what sized plate) are you preforming the isolation from?
5 petri dishes (5x 150mm plates of cells)
3) How pure do you need them to be?
as much as possible
4) What is the intended use (i.e. what quantities do you need, any how healthy do they need to remain.
it's for em 25 uL will be sufficient and healthy as well too.

thanks

I will try the classical differential centrifugation protocols.

thanks again
Bryan
Reply With Quote
The Following User Says Thank You to Nemesis05 For This Useful Post:
admin (09-06-2011)
  #4  
Old 09-01-2011, 01:23 PM
Pipette Filler
Points: 373, Level: 7 Points: 373, Level: 7 Points: 373, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Location: Grenoble france
Posts: 17
Thanks: 3
Thanked 4 Times in 4 Posts
Default Re: Intact Mitochondria isolation

I would say 5 petri dishes 25 150 mm 90 % confluency

sorry

nemesis
Reply With Quote
  #5  
Old 09-02-2011, 07:34 AM
Pipette Filler
Points: 373, Level: 7 Points: 373, Level: 7 Points: 373, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Location: Grenoble france
Posts: 17
Thanks: 3
Thanked 4 Times in 4 Posts
Default Re: Intact Mitochondria isolation

I would mean 5 petri dishes (25x 150mm plates of cells)
sorry
I really need to finalize this purification to have a good start for my post doc.

thanks
Reply With Quote
  #6  
Old 09-02-2011, 12:05 PM
obama's Avatar
Graduate Student
Points: 2,830, Level: 34 Points: 2,830, Level: 34 Points: 2,830, Level: 34
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Hannover Germany
Posts: 145
Thanks: 2
Thanked 25 Times in 21 Posts
Default Re: Intact Mitochondria isolation

Initially i used mitochondrial isolation kit. But i got better result when i used our homemade protocols and buffer. If you interest let me know (i have to look for it in my lab note).

And it will good if you write, what is the end use of this mitochondria. mean what kind of experiment you are going to perform with this mitochondria.
Reply With Quote
The Following 2 Users Say Thank You to obama For This Useful Post:
admin (09-06-2011), Nemesis05 (09-05-2011)
  #7  
Old 09-02-2011, 12:31 PM
Pipette Filler
Points: 373, Level: 7 Points: 373, Level: 7 Points: 373, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Location: Grenoble france
Posts: 17
Thanks: 3
Thanked 4 Times in 4 Posts
Default Re: Intact Mitochondria isolation

Thanks for you answer

I have already mentioned my goal
My intended use is Electron microscopy analysis of mitochondria parts
Yes I'm very interest by your homemade protocol
What kind of kit have you already use
I have use with any success the QIAGEN and miltenyibiotec kits
thanks for your help
Reply With Quote
The Following User Says Thank You to Nemesis05 For This Useful Post:
admin (09-06-2011)
  #8  
Old 09-02-2011, 01:46 PM
obama's Avatar
Graduate Student
Points: 2,830, Level: 34 Points: 2,830, Level: 34 Points: 2,830, Level: 34
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Hannover Germany
Posts: 145
Thanks: 2
Thanked 25 Times in 21 Posts
Default Re: Intact Mitochondria isolation

I used Qiagen before

Here is brief protocol

1. cells were permeabilized by resuspension in buffer containing 210 mmol/liter sucrose, 20 mmol/liter HEPES-KOH (pH 7.5), 10 mmol/liter KCl, 1.5 mmol/liter MgCl2, 1.0 mmol/liter EDTA, 1.0 mmol/liter EGTA, 1.0 mmol/liter dithiothreitol, 0.1 mmol/liter phenylmethylsulfonyl fluoride, 1:100 protease inhibitor mixture, and 0.04% digitonin.

2. Keep it for 45 min in ice (in between mix it) and followed homogenization using Dounce homogenization procedure it look like this (do five times up and down )

3. i also passed the solution using 21gugage syringe to further homogenize it.

4. Then sediment nuclei and cellular debris, cell suspensions were centrifuged at 600 g for 10 min at 4 C. The supernatant was centrifuged at 15,000 g for 30 min at 4 C.

5. After this centrification, the pellet was defined as the mitochondrial fraction.

I used this mitochondria for western blot to analysis protein trans-location experiment. but you can try it.
__________________
With Regards
Uday Kumar
[Only registered users see links. ]
Reply With Quote
The Following 2 Users Say Thank You to obama For This Useful Post:
admin (09-06-2011), Nemesis05 (09-05-2011)
  #9  
Old 09-05-2011, 08:38 AM
Pipette Filler
Points: 373, Level: 7 Points: 373, Level: 7 Points: 373, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Location: Grenoble france
Posts: 17
Thanks: 3
Thanked 4 Times in 4 Posts
Default Re: Intact Mitochondria isolation

Ok I will try taht soon
Thanks
Reply With Quote
  #10  
Old 09-05-2011, 08:59 AM
Pipette Filler
Points: 373, Level: 7 Points: 373, Level: 7 Points: 373, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Location: Grenoble france
Posts: 17
Thanks: 3
Thanked 4 Times in 4 Posts
Default Re: Intact Mitochondria isolation

I found a protocol which is adapted from Attardi and Ching (1979) and Rice and Lindsay (1997), is designed for cultured cells (2.10^8) grown as a monolayer. It's really close to your protocol. I hope it will be enough pure for my EM experiement
thanks
Reply With Quote
The Following User Says Thank You to Nemesis05 For This Useful Post:
admin (09-06-2011)
Reply

Tags
intact , isolation , mitochondria


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
mitochondria isolation lipid Molecular Biology Techniques 0 11-10-2008 01:44 PM
mitochondria isolation Vincenza Dolce Protocols and Methods Forum 0 05-19-2007 10:35 AM
ZYMOLASE Or LYTICASE, FOR mitochondria isolation lijo john IGIB Yeast Forum 0 10-04-2006 03:31 PM
Yeast Mitochondria RNA isolation Zhenglong Gu Yeast Forum 0 06-17-2006 12:17 AM
Pierce and Sigma mitochondria isolation kits sindy.loowho@gmail.com Protocols and Methods Forum 0 06-01-2006 01:47 PM


All times are GMT. The time now is 09:39 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.16961 seconds with 16 queries