Fix&Perm, biotin-SA protocols, blocking of nonspec. binding - (Feb/21/2007 )
Hi,
I have three questions I need help with:
1) Does anyone have a personal experience with Fix & Perm reagents from ADG? What protocol do you follow – could you share? I have bought Fix & Perm and downloaded some protocols from the internet, but they pretty differ among themselves. To be more specific, I need to detect both extra- and intracellular antigens at once in monocytes.
2) Could you explain in details how exactly does the FCS or BSA work when used for blocking of nonspecific binding of the secondary Ab? And how about using Fc fragments? What is it good for?
3) I have primary MAb biotin conjugated. To detect it I have ordered streptavidin-RPE-Cy5. Again, I miss some reliable protocol how to use this system. Could anybody help, please?
Thank you very much in advance.
Paja
I have three questions I need help with:
1) Does anyone have a personal experience with Fix & Perm reagents from ADG? What protocol do you follow – could you share? I have bought Fix & Perm and downloaded some protocols from the internet, but they pretty differ among themselves. To be more specific, I need to detect both extra- and intracellular antigens at once in monocytes.
2) Could you explain in details how exactly does the FCS or BSA work when used for blocking of nonspecific binding of the secondary Ab? And how about using Fc fragments? What is it good for?
3) I have primary MAb biotin conjugated. To detect it I have ordered streptavidin-RPE-Cy5. Again, I miss some reliable protocol how to use this system. Could anybody help, please?
Thank you very much in advance.
Paja
Hi Paja!
Regarding the background reduction, in our lab we have minimised the non specific binding ( flow cytometry experiments) to a greater extent by incubating our permeablized cells for 30 min with 5%BSA and do the washes after labelling with BSA containing 1% Horse serum. It was working fantastic!
Regarding the background reduction, in our lab we have minimised the non specific binding ( flow cytometry experiments) to a greater extent by incubating our permeablized cells for 30 min with 5%BSA and do the washes after labelling with BSA containing 1% Horse serum. It was working fantastic!
Hi Sruti!
Thank you very much for your tip. However, regarding the BSA, rather than a specific protocol I meant the mechanism, HOW can this minimise non specific binding. Could you explain, please? What is BSA doing so that it can prevent non specific binding?
Thanks a lot!
Paja
It binds non specifically, so it will occupy the non specific binding sites, rendering them unavailable for the antibody. so the antibody can only bind to specific sites.