I have neutravidin and want to immobilize it on 96 well maxisorp plates so i can bind biotinulated peptides to it for elisa. does anyone have a good protocol for this? what buffer, good concentration of neutravidin and peptide and so on.
Hi Gustav,
The binding protocol for a Neutravidin plate isn’t really any different from a Streptavidin protocol. For most ELISA you will generally prepare a coating buffer at a higher pH (around pH 9 or 10) to aid charging of the antigen and helping it to bind to a plate. I take it you want to use Neutravidin to reduce background/ non-specific binding? Have you tried any other methods?
A good starting point for you on your maxisorp plates would be to prepare a coating buffer as below:
Weigh 1.5 g Sodiumcarbonate, 2.98 g Sodium bicarbonate and add to 1 Litre of Water, Adjust pH to 9.6 (± 0.1).
Once you have this you can use it to dilute your Neutravidin stock. You will want to prepare your stock to 3 µg/mL and use 100 µL per well. Once you have added the coating buffer with antigen to the plate, give it a couple of gentle taps on the side of the plate to ensure the bottom of all the wells are fully immersed. Seal you plate with a plate seal and incubate at 2-8 °C for 16 hours.
The incubation time in the fridge won’t be affected if you take it out slightly earlier or later and it allows you to prepare at the end of your working day ready to start the assay in the morning.
I hope this helps to get you on your way. If you have a Neutravidin stock or kit they will usually come with some good instructions. If not, I can point you in the right direction and have added a couple of links below. If you have any questions though, feel free to ask.
Generic protocol for biotinylated assays
https://www.novusbio.com/support/support-by-application/elisa/indirect-sandwich-protocol
ThermoFisher guides to ELISA.
https://www.thermofisher.com/uk/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/general-elisa-protocol.html
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