Hello fellow scientists.
My protein is tagged with FLAG. I transfected HEK293T with either my plasmid expressing my protein-FLAG, the empty vector plasmid or the protein wild-type (no FLAG), for negative controls.
After extracting the supernatant, I filtered it and ultracentrifuged it twice as specified in Thery et al. (2006).
Then, I followed Cai et al. (2012) protocol with some small modifications. I incubated 5µL of latex beads with 10µg of extracellular vesicles for 15 min at room temp. Then I added 1mL of PBS and incubated on the shaker for 1 hour. I centrifuged at 5000g for 20 minutes (I contacted Sigma and that's what they told me). I resuspended in 100µL of my Anti-FLAG PE antibody (from Biolegend, dilution 1/100) and incubated at 4°C for 30 min, in the dark. I added 1mL of PBS and I centrifuged again at 5000g for 20 minutes. This time there was no pellet, instead the latex beads were all over the place, forming a kind of ''string''. I could still recuperate a good amount. I resuspended in 100µL of PBS and went to Cytoflex (Beckham-Coulter).
My unstained control (latex beads + extracellular vesicles, same conditions) was PE -.
ALL of my samples, including my controls which didn't have a FLAG tag, were PE + at a higher percentage than 85% (I recorded 20 000 events).
I'm at a loss. Nobody in my lab or the labs around me can help me.
Sometimes I think I should quit science and work at Mcdonalds eeerrhhhh.
Can someone help me out? I'm new in the extracellular vesicles field.
Thanks so much.