Hello,
I am working on cell lines and I make a point to validate my transfer efficiency using a Ponceau red stain, before proceeding to the blocking step. I notice that all my cell lysates show transfer after the staining, but my exosome proteins do not show anything. Does this mean the abundance of exosome proteins are relatively low in the samples or transfer has not taken place at all? Any suggestions or comments would be helpful.
Thanks,
Could you run two gels and compare the image of a gel dyed with comassie and the membrane dyed with Ponceau S?
I would add that for exosomes it is quite critical to have your proteins quantified. Thus, if you get a negative result you can be sure it was not due to insufuccient protein being loaded into the gel. One nice approach you could use that would not require much protein is what Julian suggested. You run a gel, stain it with colloidal coomassie or anything sensitive enough including in your gel a marker with quantified bands. Then, with some densitometric analysis under several considerations you can find elsewhere for reducing errors you get your stuff quantified. Some qubit kits are also available as well as bioanalyzer chips.
Best,
Daniel
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