Can anyone provide a few tips on how best to separate the protein layer when extracting total RNA and DNA from tissue homogenates? We want to try western blotting using protein from the same samples that we extract total RNA.
I succesfully did it! You just have to follow the protocol, it is simple! One suggestion: the protein pellet is like gum, I usually use the piston of a syringe to disrupt it and I wash it a lot of times. Moreover, I incubate the pellet for 30' at 37°C to facilitate its resuspension. However, it is almost impossible to totally resuspend it but the quality and the quantity of recovered proteins are good!
Dear John
Here is a protocol provided by Dr. Chomczynski (1987) who invented Trizol Kit:
The organic phase set aside during the DNA isolation step is mixed with 6 ml of isopropanol. The mixture is held at room temperature for ?ve minutes and the precipitated proteins are centrifuged at 10,000 g for ten minutes. The protein pellet is suspended in 3 ml of ethanol by vortexing, then held at room temperature for ?ve
minutes and thereafter centrifuged at 10,000 g for ten minutes. This washing/ centrifuging process is repeated three times. Following the ?nal ethanol wash, the pro tein pellet is brie?y air-dried (ten minutes) and dissolved in water. The protein yield is 6.3 mg. Western analysis are performed according to the standard protocols described in Ausbell, F.M. et al. (Eds): Current Protocols in Molecular Biology,
Wiley Interscience, New York (1990). 50ug of the isolated protein is electrophoresed in 10% acrylamide-SDS gel and electrotransferred to a nitrocellulose membrane.
Good luck,
- Badges
- Science method