Hi Cai,

To best assist you, it would be helpful to have more info such as an image of your Western blot film (or similar) w/ladder marked. Also, the running conditions of your transfer (volts and time run), your buffers, and your experimental setup e.g. are you treating the cells with something that disrupts tubulin and/or actin expression or stability?

My first question, which might provide the most info regarding your confusing results, is whether you've run a positive control (or a few). Consider including another cell line somebody else in the lab has been able to detect these controls in or run purified protein. The goal would be to have data that indicates whether it is a cell- or treatment-specific issue you are dealing with or if it is something else. Good luck.

It's almost always the wrong AB that cause it, or a bad AB. Are you sure it's AB for western? For denatured protein? For unphosphorilated protein? Does it fit your the source of your cell line (rat, mouse, human).

Another possible problem with WB newbies (although unlikely), is that the housekeeping is sometimes much smaller or bigger than the target protein, so you loose it during transfer, or don't transfer it at all.

Hi Cai,

The DM1A mouse mAb is a widely used anti-tubulin antibody that works very well most of the times. I would recommend using this Ab for tubulin detection. The fact that bands corresponding to your intended housekeeping product (e.g. b-actin) disappear in lanes where your test protein is highly detected might suggest that you probably need to pick a different housekeeping gene as a loading control. Sometimes it happens that expression of heterologous protein modifies housekeeping genes expression rates, then such genes just don't serve as loading control. I don't think you're running tubulin or b-actin out of the gels, however make sure proteins in the ladder around the size of 50 KDa are present in the blot. If so, tubulin and b-actin have to be there as well.

Good luck.