I have tried two distinct protocols for cell lysis before western blot, and have got a result I cannot yet explain. In the first protocol, cells were scrapped on RIPA buffer and homogenates were sonicated (no centrifugation step was included). In the second one, cells were also scrapped on RIPA buffer, disrupted with a 20G syringe and centrifuged 10 min at 16.000 xg and 4 °C. Afterward, both lysates were analyzed by western blot against my target protein, and surprisingly, my protein of interest disappeared with the second protocol. Equal amounts of protein were loaded to carry out the western blot. What does this observation tell you about my protein of interest?
I firstly hypothesized a nuclear localization of my target and did nuclei isolation. Unfortunately, I could not detect the protein in the nuclei fraction.
Have you stained a gel after electrophoresis (perhaps reversibly with Zn/imidazole) to check that both lysates give bands? I have had sonication screw up my proteins, but the rapid flow method is quite gentle...
Dear Dr. Buxbaum, I have stained the membrane with Ponceau, and there is closely the same band pattern in both lanes. In fact, I am analyzing two proteins, and just one of them disappears with centrifugation, whilst the other one keeps unalterable.
Dear Dr. Schmitt, it was not an actual experiment designed to test any hypothesis, but a casual observation after running gels with lysates processed by two different persons in my lab. As the protein were not detectable in one of the two lysates, we seek for differences in the protocols and found the aforementioned ones. Thereafter, we test whether it was the protocol actually interfering to the band detectability, and it was like that. As you have said, RIPA buffer should also lysate the nuclear membrane, but not DNA (as sonication does), and so we hypothesized our protein to be inside the nucleus (i.e. interacting with the DNA, as part of chromatin). However, after nuclei-cytoplasm fractionation, we could not found the protein of interest in the nuclear fraction.
We are particularly interested on what does this observation tell about the nature of our protein, as it looks to disappear with centrifugation, whilst the other target we have, does not. By the way, our protein is supposed to be completely soluble.
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