Is your selection marker before or after the IRES?

This is likely to be a dose/sensitivity issue if I understand your question correctly. The amount of protein produced by virus infection will be thousands of times less than the amount of protein produced by transfection. So after virus infection, you are presumably producing just enough protein for the cells to be selected but not enough for the immuno antibody to detect. After transfection, there will be so much protein around that the antibody would have no trouble picking it up.

Infection vs transfection is pinprick vs sledgehammer in terms of dose...

Jill Muhling.,

Thank you very much for your answer.

My selection marker is after the IRES. I have concerned about the dose, but never reali that immunostaining is less sensitive. Now., I’m also planning to perform western blot. Hopefully, western blo would resolve my issue.

Alex Kashkin.,

Thank you for the suggestion.,

Yeah I checked expressio of my protein by immunostqining before and after selection., but my luck didn’t favor me. As Jill mentioned, dosage of expression could be the reaso. If I get an opportunity I will definitely use your Transfection agent in my later experiment.

Using an IRES sequence can reduce the expression of the transgene after the IRES by 10-80%, which is why fluorescent proteins inserted downstream of IRES sequences are often very faint.

If you really need good expression then maybe consider recloning using a 2A peptide