Hi everyone,

We are trying to isolate Neuronal derived Extracelular Vesicles (NDEVs) from plasma and serum using ExoQuick and then performing Imunoprecipitation with L1CAM (monoclonal antibody, biotin, Invitrogen). The protocol seemed to work fine since we peformed (homemade) Dotblots with L1CAM as well as synaptophysin and the results showed that in the non-NDEVs no expression was detected.

However, we tried western blot under the same conditions and we can´t detect the bands. Too much backgroud, low specificity etc etc.

We have also tried preparing the samples without b-mercaptoethanol , to see whether it influenced the results.

Has anyone experience with L1CAM (WB) and exosomes?

Our conditions were:

100ug protein,

blocking: TBST 0.1% BSA 5% 1h, RT,

primary Ab: L1CAM (CD171), biotin, 1:500 in TBST 0.1%, O/N, 4ºC

Secondary Ab: Streptavidin HPR 1:2500 in TBST0.1%, 1h, RT

The weird thing is that DotBlots work fine (we repeated twice) but then again we cannot be sure since western blot is not that clear.

I would be grateful if someone could help!

Thanks a lot,