I loaded a 200ng of protein/lane for detection by WB. Unfortunately, Ponsceau S staining didn't show the proteins. What's the sensitivity of Ponsceau S? Can I procced with Western blots!?
You should. But be sure your transfer is properly done as by visualizing any marker loaded with your samples. As western can detect even 50ng or less which might not be visible through Ponceau S staining. Hope this help!
Use Pre-stained protein marker for better conformation. Because Ponceau S staining could't detect lesser concentration of protein.
Good luck
yes, one should still move ahead for western blot in such a scenario. since u mentioned that u loaded 200ng, which nearly lies in the detection range of Ponceau (supplied by sigma- 250ng or above) there is high chance you were unable to get staining.
Best
Dear Robert,
200 ng of protein is not sufficient to see bands with Ponceau. Start with 20-30 ug of protein. After transfer, insure to wash the membranes with Water or TBSTween 0.1% for 3-5 min before staining with Ponceau, because methanol and ethanol inhibit the binding of Ponceau on proteins if the membranes are not washed correctly.
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.
Good luck
200 ng is insufficient to see bands with Ponceau, but if the transfer is fine, you should see at least the bands of marker on the membrane. If not, the transfer step had any problem, probably. What are your conditions of transfer?
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