ELISA with denaturated proteins? - (Sep/22/2005 )

hi everybody,

sorry for this silly question but is it possible to coat ELISA plates directly with recombinant proteins isolated under denaturated conditions??
i've never done it before but now, we've recombinant denaturated proteins in our hands to check by ELISA and the question is, if we have to refold these proteins before using. but by the way, unfortunately we've no experience with refolding of proteins...
and if it is not possible, can someone tell me why not??
thanks in advance for your answers!

I've used denatured proteins with ELISA and it works fine.

You can peform a direct ELISA with denatured proteins. But you have to remember that not all ELISA's are the same, and not all antibodies will recognise the same protein in different conformations. If you are using a polyclonal Ab then I suspect there is a better chance of it working, or better still a monoclonal directed more towards the terminus of the peptide in question.

But you can't expect the same amount of denatured protein to behave, or produce the same signal, in the same assay.

It'll probably take a little validation work to sort it out and to see if you are getting binding of a high enough level to make sure the assay is useful.



HMilk

You can peform a direct ELISA with denatured proteins. But you have to remember that not all ELISA's are the same, and not all antibodies will recognise the same protein in different conformations. If you are using a polyclonal Ab then I suspect there is a better chance of it working, or better still a monoclonal directed more towards the terminus of the peptide in question.

But you can't expect the same amount of denatured protein to behave, or produce the same signal, in the same assay.

It'll probably take a little validation work to sort it out and to see if you are getting binding of a high enough level to make sure the assay is useful.



HMilk

thank you hairymilk for this advice. i will discuss this point in our lab. but coming back to my question. did urea interferes the assay? got anybody have experiences?
[/quote]

Well if you use too much Urea it might affect the binding of the mAb's if you don't wash carefully cause mAb's are proteins right ? and proteins will be denatured by Urea and then the binding capacity of the mAb will be affected.

You can coat directly with the Urea but I would wash very carefully that's all.

I did many Elisa with Recombinant protein (that's actually my job) but I usually dilute them in pBS prior to the coating

I use mainly Immulon 4 plates or Maxisorp at a concentration of 10 micrograms per ml and it works fine

pesji

[quote name='pesji' date='Sep 28 2005, 03:49 PM' post='26008']
thank you hairymilk for this advice. i will discuss this point in our lab. but coming back to my question. did urea interferes the assay? got anybody have experiences?
[/quote]

Well if you use too much Urea it might affect the binding of the mAb's if you don't wash carefully cause mAb's are proteins right ? and proteins will be denatured by Urea and then the binding capacity of the mAb will be affected.

You can coat directly with the Urea but I would wash very carefully that's all.

I did many Elisa with Recombinant protein (that's actually my job) but I usually dilute them in pBS prior to the coating

I use mainly Immulon 4 plates or Maxisorp at a concentration of 10 micrograms per ml and it works fine

pesji
[/quote]

hi pesji,
thanks for your answer and yes your' re right, ab are proteins. thanks for the advice with the plates and the concentration. i will try it!!!