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1 Given what little there is on the web about IARs and the negatives that can come with them, I was wondering if anyone on this forum had any experiences, tips, tricks, warning, or anything like that. For what it's worth, I'm not going to use IARs anymore, but that's mostly due to these findings. A: The current answer to your question, as of today, is: No, you can not reuse an Ig. You need a new one for each binding. (This is what the binding in question does anyway. The binding is not reversible, it is just like a diazirine-crosslink). However, in the case of your answer: It is known that a protein can have multiple isotypes due to alternative splicing. Both RARα and RXR are ubiquitously expressed, while RARβ has a more restricted tissue distribution.  These different isotypes do share some similarity, but are distinct enough to have a distinct structure and function.  In addition, it is further known that some isotypes of a protein have similar function; for example, both GAL4 and GAL11 transactivate the same set of genes, even though they are clearly different in sequence and structure. These isotypes may often or may rarely be able to participate in heterodimers with each other. When they do, they will likely both bind the same DNA molecules. The range of DNA sequences that are binding sites for a given protein could be very different from one protein to another. Think about the recognition of DNA; the way that DNA is read is often specific, but also highly degenerate. By use of the term "specific binding" I am talking about the ability to bind specifically to one protein with respect to all the others; that is, you can not bind another binding site for a different protein without degrading a large part of the DNA or creating a whole different kind of DNA. Therefore, you do not need to reuse an Ig to figure out which protein is at what binding site; a different protein from a different Ig will do. How do you know which other proteins are being tested? By their specificity to their own DNA sequences? Yes. Are there other factors, besides binding, that might distinguish the different binding factors? Yes. How do you distinguish binding

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