stable cell screen - (May/13/2008 )
I generated the stable cell lines and are now testing the cells with Western and immunostaining. Since I put a myc tag to my gene, I use the anti-myc antibody.
My western is working, I can see the band. But immnofuorecence staining is negative.
Can anyone tell me why the staining can't work? How can I make the staining work?
Can't really tell you what the problem is if you don't tell us what protocol you are following. What cell line are you using? How are you fixing your cells? If using paraformaldehyde, are you permiabilizing the cells? Which myc antibody are you using? I routinely use the 9E10 mouse monoclonal for IF and have never had problems. I even have an Alexa Fluor conjugate which makes it even easier. Are you getting dapi staining ok? Are you sure that the band you see in the western is your myc-tagged protein and not a background (ie: have you run a lane of control cells not expressing)? Maybe if you tell us a bit more about what you have tried we can help pinpoint where the problem is. Otherwise, sorry I can't be of more help.
I made the stable cell lines with 293Flp-In Trex cells. The gene is a transcriptional factor(TF) which has two alternative splice forms TF#1,TF#2. I made two stable cells: 293Flp-In Trex-TF#1 and 293Flp-In Trex TF#2. TF#1 is smaller than TF#2 and its expression level is high. TF#1 is very easily detected with both Western and staning.TF#2 can be detected with western but the expression level is low. The expression of both the genes is induced by tetracycline so I used the cells without tetracycline as negative control. The negatice control doesn't have the right band on western.
For staining, I fixed the cells with 2%paraformaldehyde 40 minutes, permealized with 0.2%triton-100 10 minutes.The primary antibody I used was 9E10. I don't think ther are any problems with the staining procedure becasue 293Flp-In Trex-TF#1 showed positive staining.