Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

which ELISA-based or western blot-based apoptosis method to choose?


  • Please log in to reply
4 replies to this topic

#1 baaran

baaran

    member

  • Active Members
  • Pip
  • 19 posts
2
Neutral

Posted 30 September 2012 - 02:36 AM

Hi all
I want to assess anti-proliferative effects of a plant extract on cancer cell lines. I should find a way to detect apoptosis. BUT the problem is we do not have flowcytometer here so we must ignore flow-based methods. Now I do not know which method to choose. Is one method enogh? How is caspase activity test, is there any ELISA-based method or Wester-based method available? How is the DNA ladder method? which one is more reliable? If u want to do the same thing, which methods u will select?
your urgent response is highly appreciated.
Thanks in Advance

#2 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 30 September 2012 - 10:10 PM

there are many different methods. I'll list them below

- CPE detection by AO/PI. this method is very easy and it's based on morphology change. You may need a fluorescent microscope.
- DNA ladder method. DNA fragmentation kits are commercially available, but you can also collect the dead cells and extract DNA yourself. You need to run the DNA on agarose gel and if it is fragmented it means the cells are definitely dead....this is the easiest explanation I can give.
- Annexin V/PI method, which is usually by flowcytometer and you don't have it
- caspase activity. This could be done by western blot or ELISA. you need to buy antibody against caspase-3 or 9 or 8. It depends really. They are commercial kits for this method too.

I would go for DNA fragmentation first.

#3 baaran

baaran

    member

  • Active Members
  • Pip
  • 19 posts
2
Neutral

Posted 03 October 2012 - 01:22 AM

there are many different methods. I'll list them below

- CPE detection by AO/PI. this method is very easy and it's based on morphology change. You may need a fluorescent microscope.
- DNA ladder method. DNA fragmentation kits are commercially available, but you can also collect the dead cells and extract DNA yourself. You need to run the DNA on agarose gel and if it is fragmented it means the cells are definitely dead....this is the easiest explanation I can give.
- Annexin V/PI method, which is usually by flowcytometer and you don't have it
- caspase activity. This could be done by western blot or ELISA. you need to buy antibody against caspase-3 or 9 or 8. It depends really. They are commercial kits for this method too.

I would go for DNA fragmentation first.


Thanks for your Detailed Resonse
I wil choose DNA Fragmentation but
I have one more Question: Selection of Caspase is dependent on which factors? which one should I Select? 8?
thx again

#4 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 03 October 2012 - 02:30 AM

You must study the apoptotic pathways. Most people check caspase 3. thst is the last caspase in both main pathways.

#5 baaran

baaran

    member

  • Active Members
  • Pip
  • 19 posts
2
Neutral

Posted 09 October 2012 - 01:32 AM

Thanks again, so I will go for DNA Fragmentation 1st and then Caspase 3 activity




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.