I am currently trying to run solubilised mitochondria extracts from mammalian cells through a gel filtration column in order to separate proteins by size & visualise protein complexes. My protocol involves solubilising mitochondria extracts (from a sub cellular fractionation) in a buffer containing 1% digitonin (10mM MOPs, 50mM KAc, pH 7). After solubilising this for 30min on ice, the solution is clarified by centrifugation at 50,000 x g for 30min at 4C. The supernatant is then loaded onto my superose 6 10/300 column, equilibrated with a similar buffer (10mM MOPs, 50mM KAc, pH 7 + 0.1% DDM).
The problem I am having is that a significant amount of my extracts is eluting at the void volume, which is >2Md for this column. After probing for proteins in this void fraction I am seeing many non-related mitochondrial proteins and so conclude this is non-specific aggregation. Does anyone have any solutions on how to prevent this from happening?
0.1% dodecylmaltoside is 2 mM. The critical micellar concentration is ~0.15 mM.
The 0.1% DDM in the column equilibration buffer may be insufficient to keep all the membrane proteins fully dissolved if the protein concentration is too high.
Another issue is that the micelle size of DDM is ~50,000. If each protein molecule were dissolved in its own micelle, all the micelles would have a molecular weight of 50,000 + the size of the protein. The ability to resolve protein molecules on the basis of their size is reduced because of the size of the micelles.
Even worse, if the detergent concentration is not high enough, there could be more than one protein molecule per micelle. The insufficient supply of micelles therefore causes the proteins to behave as an aggregate.
One possible solution is to increase the concentration of DDM relative to the concentration of protein.
However, it might be better to switch to a detergent with small micelles, such as sodium cholate or CHAPS, that will have less of an effect on the separation of the proteins by size. If my memory is correct, sodium cholate was used in the past to reconsitute mitochondrial membrane complexes into proteoliposomes, so it may be a good choice.
0.1% dodecylmaltoside is 2 mM. The critical micellar concentration is ~0.15 mM.
The 0.1% DDM in the column equilibration buffer may be insufficient to keep all the membrane proteins fully dissolved if the protein concentration is too high.
Another issue is that the micelle size of DDM is ~50,000. If each protein molecule were dissolved in its own micelle, all the micelles would have a molecular weight of 50,000 + the size of the protein. The ability to resolve protein molecules on the basis of their size is reduced because of the size of the micelles.
Even worse, if the detergent concentration is not high enough, there could be more than one protein molecule per micelle. The insufficient supply of micelles therefore causes the proteins to behave as an aggregate.
One possible solution is to increase the concentration of DDM relative to the concentration of protein.
However, it might be better to switch to a detergent with small micelles, such as sodium cholate or CHAPS, that will have less of an effect on the separation of the proteins by size. If my memory is correct, sodium cholate was used in the past to reconsitute mitochondrial membrane complexes into proteoliposomes, so it may be a good choice.
Adam B Shapiro Hi Adam, Thanks for your answer. I'll try out both of your suggestions. For your first point regarding increasing DDM concentration, I initially went for 0.1% because are you are saying it is just above the CMC of DDM. How much above the CMC would you recommend? For my initial solubilisation I am keeping my detergent far above the CMC (>16X) and keeping the protein:detegent ratio above 10:1 as well.
As far as I am aware, these conditions do not need to be as strict for keeping proteins in solution after they are solubilised? Would something like 0.2% or 0.3% be sufficient do you think?
In my opinion, sodium cholate is the better choice to prevent protein aggregation
Please look at the following below links which may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556795/pdf/ljlc35_2923.pdf
http://kirschner.med.harvard.edu/files/protocols/GE_gelfiltration.pdf
https://www.harvardapparatus.com/media/harvard/pdf/Guide+for+Gel+Filtration.pdf
https://www.chromatographyonline.com/view/protein-aggregates-and-gel-filtration-chromatography-improving-quantitation-and-throughput-uhplc-m-0
Thanks
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