No bands with Ponceau S....should I still run the Western?

Hi All:

So I just ran my transfer, and my ladder transferred all well and great, BUT when I stained with Ponceau S, I do not see any bands at all! So my question is, should I still proceed and do my primary, secondary etc? Perhaps the Ponceau is not specific enough? Have any of you ever been in this situation, and ran your western and it worked anyways?

Please let me know.  Any help would be awesome and you shall be blessed by a good luck midget =)

No but seriously, help! Thank you!

 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 your sample, and that you get decent transferred of pre-stained marker, I don't see anything why not to proceed to the blotting procedure.

If you really want to see your proteins on the membrane, an alternative way that I personally think is much sensitive than Ponceau S staining, (if you are working with PDVF membrane) is to perform a coomassieR250 (0.1% in 30% methanol and 30% acetic acid) staining. 
Briefly, Incubate membrane in 100% methanol for ~ 30sec. Then, coomassie R250 stain for 1 min. Destain in 50% methanol, 3 times, 3 min each. After that, you can use 100% methanol to remove all the staining and perform western blotting per usual.

-- Note, it won't work with nitrocellulose membrane.

Good luck.

please try for more sensitive like amido black or commassie stain if that doesnt give result i will prefer to make sample again to not waste antibodies

 I had the same thing happen twice this week.  Please help!!! What did you do to fix it? I have run many westerns before but never had this problem.  I had small amounts of protein in some lanes (exosomes) but large protein in others (4 lanes of whole cell extract).  I ran a second gel after the first one gave me trouble, with new whole cell lysate, it ran beautiful, but after transfer only ladder was present with ponceou S staining.  What has suddenly changed with my western?

Thank you so much and I desperately hope to hear back soon!