Hi everyone,
I am trying to coat microplates with HA but I am facing difficulties: in fact, if I stain coated plates with Alcian blue no or very little staining is observed.
My concerns are:
is PBS a suitable buffer for coating with HA?
what is the right incubation time and temperature? I incubate at 4°C O/N
Should I use tissue culture treated or non-tissue culture treated plates? I tried both but with no significant differences.
How can I make sure the plate coated correctely? As mentioned, I used Alcian blue (1% in 3% acetic acid).
Thanks in advance for your help, if anybody can provide a reliable, detailed protocol that would be great!
Best
Hi Carlo, coating hydrophilic polymers onto hydrophobic plastic is a challenge.
I don't know of any detailed protocols, but when I has working with heparin, I dried 100ul solutions of 10ug/ml heparin in H2O onto 96 well TC microplates.
After drying, there was significant heparin stuck to the wells.
Dear Steingrimur,
thanks so much for the reply. Actually is what I am trying right know. Adding the coat solution and air dry for 24 hours. Hope the coating will resist the furher washing steps.. I'll keep you updated.
What I find odd is the amount of published papers reporting a coating protocol for hyaluronic acid by incubating HA in PBS at 4°C O/N, just like with proteins. I wonder how many of these authors tried to stain their "coated" plates to check if the coating did succeed.
Thanks again for your helpful answer
best
Carlo
Hi Carlo, a prevailing and erroneous thought is that if you pipette anything onto plastic microplates, it will bind.
The plastic in the ELISA-competent microplates has been specially treated to be hydrophobic to allow for protein immobilization. For some companies, this treatment is a trade secret.
Agree!
thanks for your feedback! I'm having some results with the air drying protocol but after washing, the HA-bound amount is significantly poor. Keep trying,
best
Carlo
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