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
- - - - -

Indirect Flowcytometry


  • Please log in to reply
22 replies to this topic

#16 Rnotk

Rnotk

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 101 posts
3
Neutral

Posted 15 July 2011 - 09:31 PM

If I decided to use for example 1:1000 dilution of anti-fd, then I will use same dilution for all three conditions. otherwise it is hard to interpretate.

#17 baaran

baaran

    member

  • Active Members
  • Pip
  • 19 posts
3
Neutral

Posted 16 July 2011 - 03:32 AM

Maybe you are right BUT based on data sheet of the anti-fd, the amount of anti-fd should be adjusted according to the number of scFvs. Also I've tried the same concentration of anti-fd for three conditions but it didn't work...

#18 Rnotk

Rnotk

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 101 posts
3
Neutral

Posted 16 July 2011 - 10:20 PM

how about doing the experiment without M13 phage? (as negative control)
The problem you are having is the isotype gives you high fluorescent value right?

some antibody such as anti-fd or tertiary antibody somehow attaching regardless of scFv affinity.

I understand that you want to change the concentration of anti-fd according to the scFv concentration,
but if the system is working, M13 isotype should not give high value regardless of concentration of anti-fd antibody.

or simply you are using too high concentration of each antibody since you are using three antibodies and more antibodies will amplify signal quite significantly

#19 baaran

baaran

    member

  • Active Members
  • Pip
  • 19 posts
3
Neutral

Posted 17 July 2011 - 12:07 AM

I can't omit the M13 Isotype control because it is the backbone of my scFvs. I have to use it to show how much the M13 alone can bind to the target cells. "the amount of anti-fd should be adjusted according to the number of scFvs" BUT the scFvs are expressed at the surface of M13 bacteriophage. And also anti-fd is an antibody that binds to the M13 phages. So in order to exclude the nonspecific binding of the M13 phage(alone with no scFv on its surface), I must use the M13 phage alone.
And from your previous replies, i'd better to use fixative reagents before treating the cells with scFv?

#20 Rnotk

Rnotk

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 101 posts
3
Neutral

Posted 17 July 2011 - 10:30 PM

so for your isotype you have M13 that binds to the target protein on the surface of target cell.
Does this M13 binds to the target cell using scFv on thier surface or different interaction?

I thought your isotype M13 suppose not to bind target cell.
if it does, what is your experimental? not phage but pulified scFv?

The reason that I suggest no M13 is to check the system, because you are having too high background
so to me you either using too high concentration of each antibody or antibody(ies) you are using bind unexpected mannar.

#21 baaran

baaran

    member

  • Active Members
  • Pip
  • 19 posts
3
Neutral

Posted 21 July 2011 - 12:21 AM

M13 (alone) binds to the target cells BUt not in a specific manner, because it doesn't have any scFv on its surface. On the other hand, I am not getting too high background, about 15 to 20%. BUT the problem is that my test conditions show the same amount of fluorescence or less.

#22 Rnotk

Rnotk

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 101 posts
3
Neutral

Posted 25 July 2011 - 07:30 AM

then I guess something is not working,
if I were you, I will fix the sample (target cell) before staining.
capping might be the reason you are not getting signal.

if you could have some kind of positve ctrl, that would be nice though.
or you could try immunocytochemistry to see whether you can see the signal under fluorescent microscope.

#23 baaran

baaran

    member

  • Active Members
  • Pip
  • 19 posts
3
Neutral

Posted 07 March 2012 - 10:15 PM

Hi again after apx 9 months!!!
I've finished my thesis and defensed it n now that I m trying to write my article, i do not know how to discuss the negative result of my flowcytometry...
Hope u be fine n sound




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

©1999-2013 Protocol Online, All rights reserved.