Hi All,
Our lab is trying to use AlphaScreen to find protein-protein interaction inhibitors for a nuclear protein (90kDa). We thought it would be easy but our design didn't work (no binding signal at all!!). Could anyone look through our AlphaScreen design and tell us what went wrong? Also, any suggestions related to designing AlphaScreen for protein-protein interaction will be much appreciated!!
Our AlphaScreen design is as following:
Streptavidin donor beads bind to the biotinylated anti-Flag tag antibody, and the antibody binds to the N or C-terminal Flag-tagged version of our target protein.
Ni-NTA Acceptor beads bind to the his-tagged protein binding partners of my target protein (based on the literature).
The concentration of protein I am currently using is:
Anti-Flag tag antibody: 50 nM
Target protein and its binding partners: 100 nM
Donor or Acceptor beads: 10ug/ml
Buffer: 50 mM HEPES, 150 mM NaCl, 1mg/ml BSA, 1mM DTT, and 0.01% Tween 20 (pH=7)
Thank you in advance!
Lyra
I have tried following optimization and nothing seems to work..
1. Use buffer without BSA, Tween-20, and DTT
2. Change the buffer pH to 6, 6.5. 7.5, and 8.
3. Varied the concentration of both binding partners (100, 200, 300, 400 nM)
I emailed the tech support of Perkin Elmer and following is his reply. I am going to try out his suggestions and will keep you guys updated on how the things turn out :-)
"It looks like an extremely high amount of the biotin-tagged Anti-FLAG antibody-she is saturating the SA donor beads for sure. If you want to continue with the antibody approach, you should titrate it down to the 0.3-3 nM range (or 10 nM max). You should also cross titrate the amount of each of her proteins. And your order of addition is really important- you should be adding your binding proteins, the acceptor beads, and the biotin-AB, (you can vary whether you add the acceptor beads & the biotin-Ab in one step and with one incubation, or separately with longer incubation) then at the very end add the SA donors with a 30 min incubation before reading. Adding the SA donor beads at the same time with the biotin-Ab will slow things down considerably, and you may never see signal.
For PPI assays, we like to use the directly conjugated anti-tag beads and trying them in each configuration available. For example, testing Anti-FLAG donors with Ni Chelate acceptors & the reverse configuration of Nickel Chelate donors and Anti-FLAG acceptors. You could also test Anti-His beads in place of the Nickel Chelate acceptors. Buffer conditions can also be tested and optimized. What works best really depends on the proteins involved and will vary from assay to assay. I’m attaching the protein-protein interaction guide which you should also find helpful."
Hi All,
I solved my problem :-)
This time, I added only 5 nM of anti-Flag antibody.
Also, I made a mix of "antibody+two binding partners+acceptor bead" first, incubate for 30 mins. Then add the donor bead, incubate for 30 min before read.
Hope this info can help other people who face the same problem as I did!
Hi Lyra, I am having somewhat similar problem in that I see signal only at high enzyme concentration of about 400 nM and above. I would like to see signal at a lower concentration for the purpose I am developing this assay for. I have optimized the buffer to 20 mM HEPES, 25 mM NaCl and 0.05% Tween 20 (pH=7.5). Streptavidin binding partner is at 25 nM.
Did you have any insights from your work?
Thank you.
Hi Anuja,
Sorry! I just saw your posting today! Could you describe a bit more about your assay? What kind of acceptor bead are you using? Is it Ni coated? Are you looking at protein-protein interaction or protein-peptide interaction?
Could it be that the affinity between your enzyme & ligand is actually quite low and that's why you can only see a signal at higher enzyme concentration?
Best,
Lyra
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies