Can I just skip RENATURING? - Difficult native prep want to use denaturing (Nov/02/2006 )
I have a his-tagged protein that appears to be forming inclusion bodies in my preps. I have tried reducing temp. to 17C (O.N.) and adding less IPTG (0.35 mM) with little success.
I want to send this protein away for antibody production for an ELISA assay. I read in Qiagen's Qiaexpressionist (p.106) that using the denaturing procedure is fine for antibody production and that renaturing is not needed?!
Can I get some feedback on this????
WHY AM I WASTING MY TIME WITH THE NATIVE PROTOCOL THEN?! I could have been using the denaturing protocol this whole time? Man O Man.
I guess that if you want antibodies for ELISA, you want antibodies directed against the native form. So you would have more chance to get these antibodies if you immunize with a native protein that with a denatured one.
Now, if you would like an antibody for western-blot... it would be easier !
native immunogen derived Ab is beside ELISA also usable for IP; I suggest to run the renaturaization protocol;
I have raised antibodies to His-Tagged proteins which are completely insoluble under native conditions (despite refolding efforts). Go ahead and purify in a buffer containing urea but make sure the protein is of high enough concentration so the immunizing volume is low - otherwise the animal will get sick. (Check the MSDS for the LD50 and calculate from there based on the weight of the immunizing animal.)
You can get antibodies with the denaturated protein, you can get them even with just a short part of the protein. In polyclonal antibodies you have a mixture of antibodies that can either have a sequence of the protein as a target or that can get against a part of the protein with a given conformation. If you make them with the denaturated protein you will get a mixture of AB against different parts of the sequence and therefore for the ELISA probably only some of them will work (only those that are against a sequence that due to the folding of the protein is over the surface of the native protein).
So I don't know if so far it's been too hard to get it, maybe you shoul tried!!!
You shouldn't be injecting the antigen in Urea, your department ethics board will not like this?
You should either gel purify or dialyse the protein so that the antigen is in a suitable buffer before injecting into the mouse. The healthier your mouse/rabbit the better your specific immune response will be...
Holyrail, we make all our antibodies using denatured protein and the majority will still recognise the native protein, particularly polyclonal ones.