We use following buffers for dynabeads coupled with antibodies. It might be helpful;

Binding buffer (BB) (fresh)

20 mM HEPES

110 mM KAc

5 mM NaAc

2 mM MgAc

1 mM EDTA

pH 7,3 (KOH)

Wash buffer

BB + 0,5 % Triton X 100

Best

you should first block your beads before adding your protein lysate (BSA+0.05% NP40+PBS). This should help getting rid of a lot of the background

@Anukana

I think it is not good idea to block NHS-terminated beads with a protein, right?

@Erman

The principle behind blocking is that it blocks all the antibodies binding sites. Then only the protein having high specificity towards the antibody can replace the BSA to bind to it. So blocking essentially reduces the background for anything and there is no harm in trying to block the NHS terminated beads.

Also blocking the beads reduces the chances of non specific protein binding to the beads itself (as what Valeriia's concern is).

No I don‘t think so

Then NHS interracts with BSA‘s primary amines so she will have bsa in elutions

@Anukana

Erman was right before me intruding: indeed I avoid BSA blocking not to get it in my eluate (that was already tested). And the concentration of my protein is very low, so it's basically all BSA in the eluate.

Okay. I learnt something today then :). Thanks guys

Hi,

You could pre-clear your lysate with uncoupled beads. That is a good way to get rid of unspecific binding.

Kind regards,

Simon

Hi Valeriia

I was wondering if you were able to find a solution to your problem and if so what worked well for you?