Which blebbistatin derivative should I use?
(András Málnási-Csizmadia answers)
I think p-aminoblebb is a useful compound because it is highly soluble in water, non-fluorescent, stable and non-toxic. Especially its high solubility makes it very useful because its pipetting is much more reproducible compared to blebbistatin.
Nevertheless, its less hydrophobicity slows its penetration to the cell. Also, please note that its IC50 is a little bit higher compared to blebbistatin.
In summary, in many cases, especially in the in vivo experiments or when you want a longer (more than 20 min) treatment it is very useful because its concentration remain stable.
If you want fast cell penetration and lower IC50, I recommend p-nitroblebbsitatin which is also non-toxic, stable and non-fluorescent, however, its water solubility is approximatelly the same as that of blebbistatin. However, even in this aspect p-nitrobleb is much better than blebbistatin, because blebbistatin forms tiny crystals during its precipitation which completely covers the microscopic image, while p-nitrobleb produces slowly large crystals, therefore they practically do not disturb the image.
(reference for p-nitrobleb: Angew Chem Int Ed Engl. 2014 Jul 28;53(31):8211-5. Képiró M1, Várkuti BH, Végner L, Vörös G, Hegyi G, Varga M, Málnási-Csizmadia A. para-Nitroblebbistatin, the non-cytotoxic and photostable myosin II inhibitor. )
So, depending on your experiment you may use p-nitrobleb or p-aminobleb. In the in vitro cellular tests most of the time I use both in order to exclude possible artifacts.