Dear Researchers,
I am working on mitochondrial localized protein, I hereby expressed it in HEK cell line with C terminal FLAG and confirmed its targeting.
Now I want to purify to send it to be sequenced by Edmann degradation for knowing if the Targeting signal is retained or not?
Though I am confident of purifying using FLAG system, my concern is about the yield from cell lines.
For example,
minimum 1nmol of my protein around 17kda is required which comes around 20ug of purified protein.
I estimated the expression level of my protein of around 3-5ng/1ug of purified mitochondria using Dot blot.
To reach, 20ug of purified protein it roughly estimates to around 20mg of mitos minimum that has to be isolated from cell lines which I believe would be a painful undertaking.
So my final question is,
Is there any better way to purify the same in higher quantities from cell lines?
Or the yield of expression I mentioned is actually in a acceptable range or in low range?
Any advice on this question will be very helpful.
Please Note:
If nothing helps, of course I can resort to Co-IP from mice liver mitos where I have a lot. I shifted to FLAG expression for its ease than the CO-IP.
Now I dont know if I made the right decision.
Regards,
Ravi
You should contact a mass spectrometry facility (expert). The method is much more sensitive.
When I was purifying TNF alpha converting enzyme, we started out in cell lines using 20-100 liters of cells. After 6 months with no success, we switched to pig spleens, and after about 6 weeks, I had worked out a protocol to easily have enough enzyme for Edman sequencing. So I recommend the mice liver mitos. Perhaps you can do some conventional chromatography first, using Western analysis, to get a purer prep before using a Co-IP, or if the Co-IP works, then just go with that.
Hi Marcia Moss,
Thank you very much. Its a valuable information for me at this point of time.
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