I Need to bind recombinant proteins to carboxylated beads (luminex) via NH2 (amine) group, but some of these proteins contain urea (1M) and/or Arginine (25mM) in the formulation. Since these molecule will bind to the carboxyl group of the beads i need to get rid of them. Since urea and arginin are probably there to increase solubility, i fear that dialysis against PBS will result in aggregation, there for im looking for additives that reduce the changes of aggregation but cannot bind to carboxylated beads.
thanks
I will first try to remove the small molecules and see that precipitation actually happens. If it happens try adding peg or glycerol or sucrose to prevent aggregation.
Hard to tell with unknown proteins, lithium perchlorate can usually be used. Trehalose might work, tho I can't say for certain either way. I can't think of a non-amine replacement for arginine since it's mechanism is poorly understood, if you have any idea of the structure of the proteins you're working with, I'd look into the best conditions for those particular proteins. Some precipitate with low salt concentration, some with high. SDS seems viable enough tho, assuming your proteins are denatured.
Hi Rutger, if you are using EDC coupling then urea is not a problem.
Arginine is a problem because it has both a -COOH and an -NH3, which will screw up EDC coupling.
My suggestion is to put the sample through a small gel filtration column, like a PD-10, and couple it as soon as it comes out.
Same with Steingrimur, since urea is not a primary amine it won't affect the coupling. Good old salt (NaCl) almost always increase solubility while maintaining the integrity of protein structure, and is quite easy to get rid of. Unless there are reasons why you shouldn't use salt I would say start with this.
Thanks all for the anwers! Indeed Urea doesnt effect the conjucation. Oddly the Ariginnine only had a small effect the on the EDC + NHS conjugation. One of the rec. poly proteins came in DTT + 0.1%SDS buffer, this one performed best with conjugation at a higher Ph (ph 8.2 NAPO4 or sodium Bicarb buffer). In this case the assay performed best with the denatured protein, measuring ABs against several immunodominant linear epitopes. Dialysis against PBS/NAPO4 7.2 or 8.0 with or witout Urea (0.5M) didnot improve conjugation/assay performance. In the end i can directly conjugate the proteins in a higer ph coupling buffer, without the need for dialysis/or PD10.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins