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Help...purify and concentrate protein for IEF


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#1 kfszelok

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Posted 26 April 2010 - 12:24 AM

I am so NEW in protein work...I want to purify secretory protein from bacterial culture medium. So I centrifuge the culture and then filter the cell away. I cleaned up the protein using Bio-Rad Cleanup Kit. Then I used Bradford method and BSA as standard to quantify the protein. However, the OD is so low and I assumed that the concentration in the medium is too low or the cleanup process loss most protein!? So I concentrate the medium from 100ml to about 2ml using Amicon centrifugal filter device. This time I run the protein using Agilent 2100 Bioanalyzer. There was no protein inside. But when I do the Bradford method, it shows 1-2mg/ml protein. I wonder that is the Bradford method give reading correspond to the protein in the medium? since I didn't clean up the protein this time. :)

also, I have following questions:
1. If I want to run IEF afterward, am I need to buffer exchange the protein? If yes, what buffer should I use?
2. Can I use H2O as the buffer for buffer exchange for IEF? I understand that it will not be stable for storage.
3. Is secretory protein easily be degraged? Is it common to have precautions to prevent protease etc?
4. Is the broth medium contain protein that will interfer the IEF result?

MANY MANY THANKS!!!!!

#2 paramyosin

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Posted 26 April 2010 - 03:48 AM

Dear ksfzelok,
I am not very familiar with the Bio-Rad Cleanup Kit, but maybe you will need to concentrate the quantity of your secreted protein in the medium in order to obtain some signal by using Bradford assay. Be careful with BSA, some media contain this protein and it usually is in such a high quantities. I donīt know in which buffer are your concentrated protein but...if you want to develop an IEF you have to use the buffer recomended in the instructionīs device, maybe you can perform a buffer interchange with a gel filtration. I have one which uses: urea (13,5 g/25 ml); bME (0.5 ml/25 ml); acetic acid (0.385 ml/25 ml); Triton X-100 (0.130 ml/25 ml), Bromophenol Blue (0.025%/25 ml).
I would never put my protein in water, never!, at least i woul use a light buffer such us PBS or so...but, if it is going to pass to a denaturant buffer, thus, I think it is possible.
As you protein is secreted and the proteases are inside the cells you should not have any problem...but I thing that you should detect by experimentation haw stable or unstable is your particular protein.
I would run a negative control just with the media concentrated in the same grade as your protein just in case.


Good Luck!!
"Research without indebtedness is suspect, and somebody must always, somehow, be thanked." Umberto Eco




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