I am intending to purify a membrane protein which should be ~400 kDa if it forms a tetramer. My final purification step is SEC by Superose 6 10/300 column. I tried to calibrate the column with Apoferritin (443 kDa) and Alcohol Dehydrogenase (150 kDa) respectively. They were eluted at 12.3 ml and 13.9 ml respectively. Regarding to my protein, I have a 13.42 ml eluted from the column. Thus, I am not sure if this is a right position representing a 400 kDa protein. Thank you for your suggestions in advance.
It is quite common for membrane proteins to elute abnormally from size exclusion columns. The protein standards typically used to calibrate SEC columns are globular proteins, but as many proteins (especially membrane proteins in detergent micelles) have a non-globular shape, they interact differently with the column resin and frequently elute earlier or later than calculated. Your elution volume for your protein is around that estimated though and not too darastically out.
Do the fractions collected for the peak correspond to what you would expect for your protein of interest when ran on SDS-PAGE? If they do then you've answered your question, the next question is whether the product is in the tetrameric state you desire, in another oligomeric state or in a mix of states.
Really you need to run a sample of your final purified product on a SEC-MALS (size exclusion chromatography combined with multi angle light scattering) instrument, as this is unaffected by protein shape and will give you the exact molecular mass. Other options if you have access to them would be analytical ultracentrifugation or perhaps mass photometry (more difficult to use with membrane proteins in detergent, but doable and has the advanatage of being very quick and using only a tiny amount of protein). Blue native PAGE is also an option, although that would not be the method I'd willingly choose as it very difficult to do right, takes a lot of time and cannot be used for precise measurement of protein size.
Let us know how you get on!
For starter, it would be nice to have at least three standard proteins to calibrate your column.
I calculated the MW based on your elution volume and it should be ~207 kDa. That doesn't seem "not too darastically out" to me :-/
Do not forget, that you do not have only your protein, but also the micelle, which mass you have to add to your protein. So to me it would seem like your have monomer + detergent micelle.
Yongliang Zhang I'm interested to know if you've made any progress with this.
Do you have an antibody to detect and identify your protein by Western or ELISA? I think the only way to characterize your SEC experiment is to collect fractions and to identify what's in them and possibly to compare with the calibrator set.
Wolfgang Schechinger Many thanks for your explanations and suggestions. After eluted from the Superose 6 column, the protein samples were detected by Western, showing that it was the one that I wanted. Also, Native PAGE showed the molecular weight was 400 kDa, which were brilliant. So I sent the samples for cryo-EM data collection.
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