We are trying to transfer an assay based on antibody capture with strep-beads coated with biotin-peptide-antigen into the 96 well format. There are multiple ways to coat the streptavidin and people either use styrene or plasma activated styrene ("high binding") for this procedure. We found at least three possible variables, got various interesting results and still feel we would be able to optimize for ever. Therefore we would like to know, what you would recommend.
The variables are
- plastic surface (styrene, (non)activated, brand)
- coating conditions (pH, salt)
- Which source/quality of the streptavidin, e.g. for the coupling to carboxy surfaces only certain qualities are working
What is your experience. What do you suggest to optimize this process?
Neutravidin (Nav) from Thermofisher is working as well as Streptavidin (SA), is non-glycosylated and is cheaper. We used both SA and Nav at 5 microgram per ml in standard bicarbonate buffer (PH9.5). We also bound biotinylated peptides and tested sera IgG binding. We did not see any difference with or without blocking with BSA after Nav and peptides suggesting the surface is fully coated and no extra blocking is required. We always used Nunc ELISA plates (high protein binding capacity).
Neutravidin (Nav) from Thermofisher is working as well as Streptavidin (SA), is non-glycosylated and is cheaper. We used both SA and Nav at 5 microgram per ml in standard bicarbonate buffer (PH9.5). We also bound biotinylated peptides and tested sera IgG binding. We did not see any difference with or without blocking with BSA after Nav and peptides suggesting the surface is fully coated and no extra blocking is required. We always used Nunc ELISA plates (high protein binding capacity).
Why not buy sa-coated 96-well plates from thermo or similar vendors? Its industrial standard and minimizes variability.
Hi Michael,
we are coating 200 ng core streptavidin (Serva) / MTP well (Costar High Binding) in PBS pH7.4 over night.
Best wishes
Michael
P.S.: Dir und Deiner Familie noch ein schönes Weihnachtsfest!
I used plates pre-coated with streptavidin from Mimotopes to bind my biotinylated peptides and they worked good for me. I initially thought of coating myself with similar conc suggested by others (200ng) but i changed my mind after reading the process of coating. It is said that it needs several days for proper coating.
We chose Jackson ImmunoResearch (now a division of Stratech) as a source of high quality streptavidin for coating plate wells for further adsorbing biotinylated peptides. When we used polystyrere plates, we irradiated them by UV light (bactericidal lamp) for 20 min (20 cm between the lamp and the plate) just before coating. I suppose UV irradiation causes the formation of a certain amount of free radicals and active oxygen-containing groups that readily covalently attach the protein that is coated on.
If you want to save Streptavidin, you can biotinylate BSA, coat it on plastic and then coat Streptavidin. This two-layer coating works very well. I don't have concentrations to suggest, you will have to try.
Hi, could some body please detail the protocol regarding streptavidin coating plates. I mean incubation conditions/ amounts per well density... Thankx
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