denatured antigen for elisa - (Jun/08/2007 )
hi all,
i am trying to find out some conformation specific monoclonal antibodies against the protein.
for that i want to denature the protein to coat the elisa wells and compare the od with that coated using native protein. i tried heat denaturation at 95 and also at 65,60,55,45 and 40 but in all cases there is precipitation of protein. can someone please suggest me methods to denature the protein and coat the elisa wells so as to detect conformation specific monoclonal antibodies
thanks in advance
leelaram
i am trying to find out some conformation specific monoclonal antibodies against the protein.
for that i want to denature the protein to coat the elisa wells and compare the od with that coated using native protein. i tried heat denaturation at 95 and also at 65,60,55,45 and 40 but in all cases there is precipitation of protein. can someone please suggest me methods to denature the protein and coat the elisa wells so as to detect conformation specific monoclonal antibodies
thanks in advance
leelaram
denaturing detergent such as SDS or DTAB (dodecyltrimethylammonium bromide) may be useful; if detergent disturbs coating, dialyse or use spin columns (f.i. Pierce) to get rid off detergent
hi,
u can use pH-2 solution to denature u r protein and after incubation neutralize it before going for ELISA.
but check out the renaturing capacities of your protein of interest.
sravan
hi all,
thank you for your valuable suggestions
i am going to try it out
thank you again