Antigen denaturation - (Nov/23/2007 )
Hi all, this is my first post in the forum. I would like to denature a antigen (glycoprotein) to compare its presentation vs. the normal form by APCs, so I need to get the denaturated form in a buffer not harmful for the cell, cos I will incubate the cells 24h with the denaturated/non denaturated antigen. I have though in heat denaturation but I am afraid that along 24h incubation the protein can be refolded and I have no idea about chemical denaturation methods. Could anyone supply me a standard protocol to do this?
Cheers
Cheers
denaturation can be done with harsh pH, sonification, detergents, heat etc;
but what do you really need? a denaturized protein or an inactive protein? may be deglycolization prevent binding to the cell surface as the glycosidyls of your antigen may interact with the glycocalyx...
What I really need denature the protein to compare its effect with the native protein
Thanks
Cheers
denaturation can be done with harsh pH, sonification, detergents, heat etc;
but what do you really need? a denaturized protein or an inactive protein? may be deglycolization prevent binding to the cell surface as the glycosidyls of your antigen may interact with the glycocalyx...
When we do cell-free assay, we also boil antibody for our control. Our experiment also take about 1h. So I think you can boil your protein to denature it. I think that it will stay killed.
Heating with or without a reducing agent, such as DTT will denature most, if not all.