I was curious to know what types of problems that users are having with various biosensors as far as coupling, reproducibility or availability. I saw a post recently about capacity issues when coupling biotinylated protein and getting less than optimal results (less than 4:1 activity) and it got me thinking about how to optimize some of these chemistries. What issues are you having? What is the sensor type and who makes it? Is there a chemistry that is not available that would be helpful to you?
Most issues are about the pre-concentration - covalent binding and the amount of active ligand after immobilization. Sometimes big discrepancies are observes, like high pre-concentration - low immobilization or high immobilization - low active ligand.
I most cases researchers are following the standard protocol (what is fine) but fail to see that some ligands (or all) need some optimization for the best results. This means that you should immobilize the ligand in different densities, use at least two different chemistries or reverse ligand and analyte. In general, the cost of a sensor chip is probably the limiting factor because users do not want to 'waste' chips. They fail to see that the investment in chips can give better results.
Bottom line is that the chemistries and immobilization strategies (covalent, capture with tags or antibodies) that are available are not used to the full extent.
I would have to agree that there are a lot of users out there that do not understand the capability of the available biosensors. I believe that this comes from using SPR to characterize an interaction system of interest instead of also learning the capabilities of the instrument too (instrumental analysis). For me, my job is to manufacture and develop surface chemistries for biosensors, so I am heavily biased towards chemistry performance instead of interaction analysis and have run many assays to optimize and characterize these types of biosensors. But I am curious. Currently, there is a big demand for 6x His capture chemistry, but before that people were exploring DNA tags (hybridization), and before that Biotin/Avidin conjugates. During this time, there have been many other various tags that we have dealt with that didn't make the cut, but is there some other tag or capture approach that isn't being used because there is not a chemistry available for it? My goal is to make ligand coupling and reproducibility as easy as possible, and I am trying to get an outside perspective on this issue.
Is there a chemistry that targets only the amine- or carboxyl-end of a protein? Because proteins in general contain several lysines, the standard amine coupling is prone to random immobilization. When you have a protein without a special tag this would be a nice option.
What is your opinion the best chemistry to pacify the unreacted activated groups on a CM5 sensor chip? Ethanol amine is normally used but are there other (and/or better) alternatives?
Thanks for the reply, and sorry to get back to you so late.
It would certainly be difficult to target the N or C terminus outright for proteins without blocking the Lysine residues as you mention. In fact, some proteins are affected by random immobilization since this may compromise the binding site, and may be where your first question was going. I had been working on an Aminated chemistry, but it is so reactive currently that it is hard to use, although I may have a solution to this issue soon. This still would not address targeting the end terminus, but depending on what groups were affected on the protein, could prove useful where Amine residues were compromised.
As for the second question, Ethanolamine is an excellent reagent due to its fast diffusion and low cost. Tris is an alternative, but I wouldn't say that it has any advantage over the Ethanolamine. One potential thought though would be the use of a short Amine-PEG reagent. PEG acts as an ion pump, so in certain applications, it might be helpful to lower the diffusion boundary (of ions and perhaps small molecules) but this is just theoretical at this point. A potential application of this could be the study of cell membranes where ion transport is needed. The use of PEG around immobilized cell membrane could facilitate the transport of ions around the membrane (provided that it was coupled in a 2-d format).
Thanks for the input; it definitely has me thinking!