Does anybody know of a good protocol to extract proteins from rat aorta? I want to see the expression of endothelial proteins such as eNOS. The biggest problem is the amount of tissue I would need as I want to do another analysis using the aorta of a same rat. I intend to use the thoracic aorta.
I used to perform western blot analysis on aortic ring (4-5 mm lengh) from rat by using simple extraction buffer such as PBS + detergent such as Tween (0.1-0.5%)+ cocktail of anti-protease. RIPA buffer can also be used. Imprtantly, I used small volume of buffer (100 µl) together with mechanic extraction with very small potter on ice. To improve extraction, you can also sonicate the preparation after potter extraction. With such method I was able to perform several western-blot (at least 10) by aortic ring. I case of mouse aorta, I recommend to use the whole aorta for western blot or aortic ring for immunohistochemistry/fluorescence.
We did a small experiment to determine the effect of RIPA buffer concentration and motion on the protein content in a homogenate. After homogenization in1x-2x-3x RIPA buffer, we put an epp with the homogenate in a cart wheel to ensure optimal effect of RIPA on the cells. We compared this to 1x-2x-3x RIPA stationary, on the bench. Protein content was measured using a BCA protein assay (Pierce). The most protein was measured after 2 hours of shaking on ice in a 2x RIPA buffer solution (with protease inhibitors). Although this answer is perhaps a bit late for your specific question, maybe others involved n similar research can benefit.
When you're looking at enos uncoupling, all procedures should be performed ice-cold, possibly in a cold room (4°C).
Jan
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