I am going to do a native-PAGE to see if my multisubunits protein (human RNase H2) is purified as a complex or if it breaks apart during NiNta purification.
I would like to know if the conditions in which my protein is running in the native-PAGE are suitable with my purpose.
If someone has a good protocol to suggest, please let me know!
Thanks a lot.
Hi Silvia,
Firstly, you can have a look at the prep coming out of the Ni-NTA column on a denaturing gel. What band pattern do you get ? As to the native gel, you won't know the answer until you try. As a first experiment, I would try to run the prep on a low-percentage (7 %) polyacrylamide gel using the Lämmli buffer system but leaving out mercaptoethanol + SDS and not heating the samples. Stain the gel briefly without fixiing (no TCA). Depending on what you get, you can cut out the bands, equilibrate them in Lämmli sample buffer (this time with SDS + mercaptoethanol) for 30-60 min and then loading the pieces of acrylamide on a second SDS-Page gel to identify the components. The gel pieces should be covered with sample buffer before starting the electrophoresis. In order to enable the insertion of the gel pieces into the second gel, the second gel should be cast with thicker spacers. Alternatively, you could also cut out a strip corresponding to a lane of the first gel and layer it on top of the second gel. The components of each species present in the first gel will then appear as spots in the second dimension
It's appropriate ... You can also do one thing. SDSPAGE. In one lane sample plus non reducing sample buffer and in one more lane sample plus reducing sample buffer.. If both lanes show same pattern then it means that the protein is no more in complex state. If first lane is single protein and second lane has 2-3 subunits (based on the complex protein) then it means protein has survived and maintained its complexity.
Best
RRA
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