Regarding conjugation of a HyNic peptide and 4FB oligo… we are mostly concerned with getting all of our 4FB sites on our DNA conjugated to protein, not with the efficiency of protein conjugating to the oligo. The instructions for conjugation seem to suggest a ~2:1 ratio of DNA to protein to obtain 95% protein conjugation. But, if we are more interested in the DNA, then I assume our ratio should be less. I am wondering how much less? Also, if we would like to store some of the HyNic peptide for later use, is it stable if stored in buffer at –80ºC?

If your main goal is to conjugate all of the 4FB oligo, then you may want to use a ratio closer to 1:1. The number of oligos a given protein can carry is largely governed by its surface area, which is roughly proportionate to its molecular weight. In our experience, an antibody has no difficulty conjugating to multiple oligos in the 20–30 nucleotide range. Smaller proteins would do better with a 1:1 or perhaps 2:1 DNA:protein ratio. Particularly, if you don’t mind having free protein in the crude reaction, then this is not a problem. I would add our 10X TurboLink catalyst buffer to drive the reaction to completion. Regarding the 4FB on the oligonucleotides, we order the amino-derivitized oligo and then incorporate the 4FB post-synthetically using our 4FB HNS linker. This process is quantitative in our experience. The ultimate purity of the oligo (in terms of full-length 4FB-modified) is therefore dependent on the incorporation efficiency of the amino linker. Therefore it is important to remember that although you might be adding 2 molar equivalents of oligo to your protein for conjugation, you’re really adding only 1.1 equivalent of 4FB oligo. The unmodified oligo cannot conjugate and will remain in the conjugation reaction. Regarding the peptide, it is most stable as a lyophilizate. If you have an analytical balance you can weigh out 1–2 mg to dissolve for your studies and store the rest lyophilized at –80ºC. Barring that possibility, you may dissolve the whole tube and aliquot it out to usable portions and store at –80 ºC. Avoid freeze-thaw of the peptide as this will cause it to precipitate and may affect the HyNic group adversely.