Guest Chiara SC Posted April 17 Report Share Posted April 17 Hello! I am using Fluorospot Plus IFNg, IL22, IL17 to detect PBMC response to viral and CNS-derived peptides. Negative responses are quite expected (eg columns 9-10 and 12), but in one particular stimulation (MOG, columns 10-11, rows A-F) I see a weird halo signal, and sometimes patchy signal in the centre of the well. What could be the cause of this? It really seems peptide-dependent, even though it has been resuspended to the same concentration (1ug/ml) and with the same buffer (sterile water) of other peptides used in the plate. Any input will be appreciated! Quote Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted April 17 Report Share Posted April 17 We know from experience that certain peptides can interact in a strange way with the PVDF membrane and cause super-weird backgrounds. We hypothesize they get stuck to the PVDF and detection reagents stick to these structures for some unknown reason. If the peptide is in very high concentration of DMSO this can also lead to weirdness in some cases. Many times you will need a high concentration DMSO to initially dissolve the peptides, but from there you can dilute it down to reasonable concentration which will not cause issues. Do you see the same membrane background for IL-22 and IL-17A? Quote Link to comment Share on other sites More sharing options...
Guest Chiara SC Posted April 17 Report Share Posted April 17 Hi Christian, thank you for your quick reply! We indeed use H2O instead of DMSO to dissolve peptides to avoid issues with the membrane (colleagues' suggestion) I do see the same background in the other channels, with the same pattern (brighter in 550 than 640, attached) Would you think it is molecule- or preparation-related? In the second case I might ask the company for another batch of the same peptide, but if it is related to the aa sequence that would not help. Many thanks! Quote Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted April 23 Report Share Posted April 23 Since the background artefact is also present in LED550 and LED640, it is unlikely to be a something connected to one of the detection mabs. I would say it is likely to be a molecule issue and producing new material wont help. But maybe you should try dissolving them in DMSO and then diluting them? Water might not be a good media for dissolving certain peptides. As long as the final DMSO concentration in the FluoroSpot well is around 0.2% its fine. You can start with a higher DMSO concentration if the peptide i hard to dissolve, and then lower in by diluting afterwards. Quote Link to comment Share on other sites More sharing options...
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