I am currently using a human IgG primary antibody and a Alexa647 anti-human Fc secondary antibody for the staining and do not see a signal for the low-abundance target.
Thank you very much!
Amplifying the signal is not a very good idea in case of low-abundant proteins. You need to understand that, the signal amplification happens to the background or non-specific binding also. However, you can find the following articles that discuss the signal amplification for flowcytometry.
http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0320(19990201)35:2%3C176::AID-CYTO10%3E3.0.CO;2-S/abstract
http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0320(20000501)40:1%3C81::AID-CYTO11%3E3.0.CO;2-K/full
https://www.thermofisher.com/de/de/home/references/molecular-probes-the-handbook/ultrasensitive-detection-technology/tyramide-signal-amplification-technology.html
http://www.perkinelmer.de/lab-solutions/resources/docs/APP_FlowCytometricDetectionpAKTandFoxP3JurkatTCellsUsingTSAPlusSignalAmplificationTechnology.pdf
I hope this information is helpful to you.
If you really stick to the plan of amplifying the signal, you must make sure that the antibody binding is highly specific without any non-specific binding. You may have to use controls for every step to detect the nonspecific signal. Goodluck
I would like to suggest you to use Tamara suggestion. PE has very high quantum yield and will result in strongest signal for the low abundant markers. See the dilution of the antibodies. Go for labelled antibody and normalize with respect cell number.
In case you want to stick with separate primary and secondary antibodies, give overnight incubation with primary antibodies at 4oC .
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