I'm using VHH nanobodies coupled to NHS-terminated magnetic beads to capture my protein of interest. However the background proteins in my eluate is quite an issue. What can be used as a wash buffer to get rid of the sticky background proteins? Currently I'm using PBS (suggested in the protocol), recently started to add 0.1% of NP-40 but it doesn't seem to help the issue. P.S. I have a funny feeling that these are not only protein-protein interactions, but the bead surface itself (silica) seems to attract unneseccary proteins. All the active NHS groups are supposedly blocked with ethanolamine.
Thanks in advance!
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.
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?
Hi Valeriia, sorry for the late answer. Blocking with ethanolamine (quenching) seems to be okay; we've done it several times with similar reactions like you described.
After that, blocking with milk powder or BSA is possible; wash thoroughly!
Sometimes silica can be the problem, as the surface is somehow hydrophobic, and it contains pores, which can be filled with non-specific binding protein.
A solution for this problem can be NHS-activated agarose or mag beads, basin on agarose. Here non-specific binding is definitely reduced, compared to silica and organic polymers.
My company sells it:
https://cube-biotech.com/products/magnetic-beads/activated-magbeads/nhs-activated-magbeads/251/purecube-nhs-activated-magbeads?c=3
But you can easily find comparable products from other companies.
Best, Roland
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