stable cell line generation - (Nov/18/2009 )

I am trying to generate stable expressed Myc-tagged protein in A549 cell line using G418 selection. I pick up 6 of single clonies after two week selection and screen Myc expression leveles by immunoblotting and immunofluorescence microscopy. I couldn't pick up Myc expression by Western. However, I can detect Myc by IF microscopy. Is it possible? Could anyone give me some suggestions why it can happen?

yxz98 on Nov 18 2009, 06:39 AM said:

It may be a sensitivity issue in IF vs Western, if you have low expression of your myc-protein.

Thanks for your response. However, from general speaking, i though WB is more sensitive than IF, isn't it?

yxz98 on Nov 19 2009, 11:09 AM said:

Not sure about sensitivity, however, I think your problem might be in the antibody not detecting reduced protein. Check the data sheet of your Ab to confirm it works both on IF and WB, because some antibodies will not detect reduced-denature protein.

Western blot using HRP and any modern ECL substrate should be about an order of magnitude more sensitive than fluorescence.

Did you have a positive control for you Myc tag antibody on the blot?

warsel on Nov 24 2009, 12:11 PM said:

Warsel, what reference do you have for such a definitive statement? If this were true, why the current push from biotech towards using flourescent Western blotting kits, touted as being the most sensitive method for Westerns? The reality is that there can be no such general comparison for sensitivity of Western blotting vs fluorescence as each is entirely dependent on the sensitivity of a given antibody for its epitope and the accessibility of that epitope in native vs denatured targets. As not every antibody can be used for both assays, I would think this would be evident. Comparisons between the two assays using different antibodies for the same target are invalid. Fluorescence is highly sensitive, though again, sensitivity also depends on your particular microscope's capabilities and settings. I have used many antibodies which are readily able to detect expression through immunocytochemistry that fail to detect the target in Westerns and have often seen flourescence from GFP that appears very bright but elicits only weak staining on Westerns.