I have plates coated with antibodies which are able to capture RNA-DNA hybrids and I would like release them for further analysis. How could I do that?
It depends on coating methods used by manufacturing company. The always try their best to reduce the chance of decoating of antibody by some patented materials, so if it a commercial kit, ask company for help.
Otherwise, some eluting buffer will work to separate ab from surface but with some damage on protein structure of ab.
we'll, you need an eluting buffer with strong ionic strength to disrupt the complex between your interested molecules and antibody. Again, you could,find,the shortcut by asking form the company.
However, since I just want to capture the hybrid RNA-DNA. I was thinking would be better release break the binding of antibody and hybrid or by a buffer (ph~7.4) or concentrated NaCl or temperature. Do you guys agree?
I'd try the same agents which usually are applied in affinity chromatography to break the antibody-antigen interaction: glycine @pH 2, >3M guanidium salt etc.
It may be simplistic but consider what is happening at the molecular level. The antibody is binding the antigen (DNA/RNA complex) ionically with other forces such as Van der Waals acting when the molecules are sufficiently close. Essentially therefore the binding process is dependent upon the ionic strength of the buffer in which the reaction is taking place. Hence to release the antigen it is only necessary to change the ionic strength of the buffer hence increasing the NaCl concentration will release but what will the damage be to the DNA/RNA? As you also indicate that you do not need the antibody you may consider using urea which will denature the protein and release the antigen. After either process you will need to confirm the structure of your antigen is retained. Something you will have to do whichever of the answers sent to you you choose to follow.
Thank you very much for answering. Exactly, it is hard to know the damage to the hybrid. I also was thinking about to try RNAse H to release the antigen antibody. Do you think it may work?