Hi all,
I recently ran a western blot using medial prefrontal cortex samples from PN17 rats. I followed the same protocol that other students have used in our lab (we prepare samples with RIPA buffer + phosphatase and protease inhibitor), and I see this weird double band where spinophilin (green) should be. I loaded 20ug of protein, and my primary antibody concentration for spinophilin was 1:1000. The red is stained for GAPDH at 1:3000. It's weird because with the same amount of protein loaded (and same protocol), in the cerebellum and POA, we don't see the double band. But we also see this double band in the amygdala as well.
Is this simply a regional difference and therefore I need to change the amount of protein loaded? If not, does anyone know why this might be the case. I'd also appreciate any suggestions to improving the method.
Thank you!
One could be modified (phosphorylated, acetylated...) . The real question is if you had pure spinophilin and preadsorbed the antibody, do both bands disappear (or if there is a knockout available). Like any other immunocytochemical test, if your detection is sensitive and the antibody concentrations are too high, then you will unmask cross-reactive bands. What happens if you dilute the primary?
It looks like your secondary antibody is cross reacting with your samples. Stain it with only the secondary antibody and see if you western looks the same. You may also be overloading your gel with 20 ug. In many cases proteins will run differently if you add different amount of protein. Try running BSA and or your molecular weight marker side by side and see if each protein runs to the same position in each. If they don't it means your system is sensitive protein concentrations.
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