I'm currently expressing and purifying a lectin in E. coli BL21. To test whether the lectin has been successfully purified, we did a hemagglutination assay. The lectin is supposed to be a positive result and it indeed appeared to be so. However, our negative control, with 50uL trypsin-treated rabbit erythrocytes (2% in 0.9% NaCl) turned out to be agglutinating.
After searching on the internet, one book suggested to add 0.1% bovine serum albumin to the saline to prevent auto-agglutinating. Does anyone know why would that be? Or has anyone had such experience? Thank you!
Albumin is typically used for such purposes. It is used in blocking of plates for ELISA as well. And why? It is cheap, well available, well characterized and it serves as an transport protein in the body so it has good ability to make interactions with non-endogenous proteins and drugs. Function of albumin and its interactions are well written in e.g. wiki:
http://en.wikipedia.org/wiki/Serum_albumin
Hi Peter, serum albumin is non glycosylated so it will not affect lectin studies.
As Miroslav said, BSA is widely used to block non-specific interactions. Just be sure to use pure preparations of BSA that do not contain plasma glycoproteins.
Thank you Steingrimur. The thing I don't understand is how BSA interacts with erythrocytes and how such interaction prevents the auto-agglutination. I tried to find anything on this, but most sources I found did not really explain the molecular mechanism behind it.
Hi Peter, BSA can block direct contact of RBCs, but in theory, trypsin treated RBCs should not agglutinate in response to lectin.
Have you tested the trypsinized RBCs with commercial lectins to see if they agglutinate?
Another thing is that RBCs can coalesce in the presence of divalent cations. (I will have to find those papers). BSA can also bind metals really well.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques