Hi scienceonly1, Have you considered cleaning the water bath for warming up your media/buffers for cell culture? Also, UV treated your hood + using proper pipets (those with fiber at the top) should help to prevent contamination too. Good luck.

Hi gvika, What kind of protein you are trying to detect? It is a protein that is expressed at extremely low level or otherwise? Have you recently gotten a new tube of secondary antibody/opened a new box of ECL? And what kind of ECL you are using? It may sound counterintuitive, however, too mu ...

Hi laumv, What kind of method did you use to visualize the final result? (X-ray film or a camera?) If you were using an ECl method, you should be getting black bands on a clear background but not the other way around. Detecting white bands on the black background using ECL based-detection ...

ankan, In addition to all the experiments that Suola suggested, you can also try a far-western analysis since you have both proteins purified. A concept of the far-western can be found in this link. Good luck.

Hi, Ponceau S indeed is pretty insensitive. The question is, what kind of sample it is ?(whole cell lysate? chromatography purified protein? IPed samples?) One will only see strong staining if significant amount of protein is transblotted onto the membrane... If you are sure you have protein in ...

Oops, sorry. I wasn't thinking about the assay part. Obviously heat-inactivation won't work. As for now, the only way that I could think of is to use some sort of chromatography to separate them.... Now, we need to know a few more info- For example, What is the Mw of your protein of interest ...

Hi calandino77, Boiling (100deg) for 10 to 15 min will do. Good luck.

Hi RAD, Have you confirmed whether you are plating the transformed E. coli on the correct antibiotic containing plate? Almost all of the destination constructs that I work with carry Amp resistant gene. Also, be sure you follow the transformation protocol "precisely" come along with ...

Hi xzheng, Either Endo H or PNGaseF only released N-linked glycans. The band shift observed on your protein might also contributed by complex O-linked glycans. (If you already have ruled out the possibility of protein isoforms from alternative splicing?) It is not uncommon that same protein homo ...

Hi calandino7, Due to the microheterogenuity commonly found on glycosylation, I am not surprise that your de-glycosylation/western blotting approach turned out unsatisfactory. That said, MS-spec approach can provide a much more convincing result.... You can find the method for PNGaseF/O18 la ...