Good afternoon everyone,
I am a master student and I recently generated a U2OS cell line carrying the fusion oncogene EWS-FLI1 (it's a doxycycline inducible system) and I would like to test via Western blot whether my cells, upon addition of dox, express the fusion oncogene. Unfortunately, I wasn't able to detect the protein yet. I have tried following antibodies:
- Polyclonal mouse anti-EWSR1
- Mouse monoclonal anti-FLI1 (F12) sc-365294.
Does somebody know an antibody that 100% works for detecting EWS-FLI1 via western blots? I am asking since I don't want to risk to buy an antibody that might not work.Thank you all in advance for any reply.
Hello Henrico, detecting EWS-FLI1 by Western blot is tricky, most commercial antibodies recognize either the EWSR1 or FLI1 portion alone and therefore also pick up the corresponding wild-type proteins, leading to weak bands. Unfortunately, there isn’t a single antibody that universally and reliably detects the fusion across all contexts. Maybe you could try: FLI1 antibodies, FLAG/HA tagging, or RT-qPCR with fusion-specific primers as an alternative to confirm induction.
Unfortunately, there is no “100% working” commercial antibody. If your aim is simply to confirm inducible expression, I’d strongly suggest combining a general FLI1 antibody Western (looking for the higher MW band) with a junction-specific RT-qPCR. If you need clean protein detection, an epitope tag is often the most practical solution.
- (PMID: 20827386; PMID: 8516324)
The answer to this question is provided by MedChemExpress Technical Support.
Hello Henrico Frau ,
I agree with the above: Most peaople use FLI1 and look for a signal at the specific height/weight of the fusion. Depending on your cells you will also see the WT at a different MW.
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publication