Posted 15 July 2011 - 09:31 PM
Posted 16 July 2011 - 03:32 AM
Posted 16 July 2011 - 10:20 PM
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
Posted 17 July 2011 - 12:07 AM
And from your previous replies, i'd better to use fixative reagents before treating the cells with scFv?
Posted 17 July 2011 - 10:30 PM
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.
Posted 21 July 2011 - 12:21 AM
Posted 25 July 2011 - 07:30 AM
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.
Posted 07 March 2012 - 10:15 PM
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