I want to conjugate anti - CD9 antibody onto magnetic bead (diametre: 7 nm ). I used 10% BSA to block unspecific protein binding after conjugation. But the results showed unexpected adhesion from non-target proteins from serum. I guess 10% BSA is not appropriate in this case. So what should I use ? Thanks very much!!
You should quench the beads after conjugation (depend on conjugation method). You can try other sera bovine, horse, calf. There are also free protein commercial blockers. Try to add some detergent like triton or tween.
Completely quenching the conjugation reaction is necessary. 10% BSA seems a bit high usually a 0.5%tween +1-5% BSA is a good place to start. Also, I don't know what your sample is or how you are detecting appropriate binding. Both of these will affect what blocking agents you use. Assuming you are sampling serum then you may want to try a small amount of Triton for problematic background binding. You will need to titrate it as it doesn't take much to mess up the binding you want to see. This has sometimes worked for me.
Dear Wang Xuchu
I do this extremely often.
first you should activate the NHS groups on your beads with HCL. are you doing that?
how much antibody are you using and what type of beads?
the antibody and anything needs to be amine-free. so if you have Tris buffer in the antibody, you need to remove it first.
After conjugation of the antibody, you need to block the still reactive NHS groups. You can use anything with an amine groupd to block the beads. I usually prefer ethanolamine because it makes the beads a bit more non-sticky. But you can also use Tris or anything with a NH group.
You should NOT use BSA, as this leads to BSA being bound to the beads! and then you have 2 problems:
1) anthing binding to albumin will bind. and many proteins bind to akbumin
2) the BSA is usually denaturated and has a high non-specific binding
after you blocked all you NHS groups, you can try to coat the surface to further reduce non-specific binding.
You should use high grade human albumin, like from clinical bottles. That is usually the best. Just use 2% in Tween-PBS. this will coat the surface. then add the Serum directly to this blocking solution. that is very important because if you have something the serum which binds to the blocking solution, it will not bind to beads directly, but to the proteins which are in solution. that also helps.
after the incubation, the washing steps are important. you should check at which pH and which salt concentration etc. you potein gets of the beads. after you did this, use a washing buffer with a higher pH to get rid of low affinity binders...
Dear Oliver,
your protocol really works fine in our hands and helped us a lot.
Cheers Janko
many thanks Janko Brand :-)
and cheers to BioTez! I often ordered your excellentt products when I still worked at the MDC. was the best time there.
Thanks for using our products; we hope we can keep you as a customer also in the future and your career after the MDC.
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