Hi all,
I study the release of gentamicin from bone cement pellets immersed in PBS. So I have been using plain 1x phosphate buffered saline as a blank and I treat it the same way I treat my samples - that is derivatize using o-phthaldialdehyde (OPA) and isopropanol. OPA reacts with amine groups in gentamicin to form UV absorbing isoindoles but I see high absorbance at 332 nm even with plain PBS which has no amine groups or gentamicin. My only suspicion is that the PBS (Fisher) i'm using may have traces of Tris or amine groups but the packaging doesn't say so. Has anyone else encountered this problem? any thoughts/suggestions?
Thanks in advance.
Is the water your are using for the PBS pure? I worked on a project where we were using OPA for amino acid derivatives and found a high background due to the organic content in the water that went through a deionization type purifier. We changed the cartridges and sterilized the water purification system and this brought the background down again. If in doubt use HPLC grade water purchased from a reputable vendor. We also found we had to work with OPA away from areas where people were doing PAGE-SDS since they use Tris-Glycine buffers at high concentrations. It seemed to us that airborne aerosols from their work easily got into and contaminated the OPA reagent and our samples.
Also, is the OPA pure? Have you tried OPA from more than one lot or source?
Thanks for your reply. I tested with deionized water that I use to make PBS and it seems to have some absorbance too although lesser than PBS. No I haven't tried using OPA from another source. I use a plate reader - that could also contribute to some background noise.
If the deionized water is contaminated with mold or bacteria the PBS can promote the growth of these organisms. Are the plates you are using made for fluorescence assays? Has the plate reader you are using been maintained or cleaned recently?
I use UV transparent plates that are made for measuring absorbance in UV range. The reader is a different lab's and its used a lot for bacterial growth assays. I'm fairly sure its maintained well although not sure about the frequency of calibration.
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