we use cell protein (50 ug/ul) to screening a series of antibodies (..., and b-actin), the primary antibody dilution according to manufacturer's instructions. and diluted the secondary antibody by referring to previous studies... but ,here the problem is that b-actin get a standard band detected. But some of antibodies no band detected(Ikk a/b), while the size marker line detected... can anyone tell me what the problem is? thanks
Hi Huan,
First thing I would ask you is about the expression of your proteins of interest in the tissue that you are using for WB. If somebody else has reported them before, that's ok.
Second, since you are loading a high concentration of protein and you are detecting the b-actin, I think the antibodies are the issue here. Review all the antibodies datasheets and be sure that they can be used for western blot.
Third, what dilution are you using for your primaries antibodies? That could be another factor affecting your results.
Fourth, I agree with Priscila's suggestion regarding the Ponceau to be sure that your proteins of interest are in your membrane.
Fifth, what is your exposure time? some antibodies require longer time than others.
Hope this information can help you.
Good luck!
Indeed the ponceau control is essential: any air bubble entrapped at the place you expect a band will prevent detection. The Ponceau also tells you that you have sufficient protein loaded in the lane and comparable amounts across the lanes. I attach our troubleshooting guide for further advice.
Good luck.
Priscilla, Ponceau S will work fine with PVDF membranes as well. I also use amido black to stain my membranes although it leaves a faint blue stain after destaining that isn't compatible with IR secondary antibodies.
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