We have a cellulase crude protein mixture with oligosaccharide contamination. I have tried various protein purification methods like Ammonium sulfate precipitation, Column based, TCA method, Ethanol and Acetone. But since my protein concentration is very low (15ug/ml) , none of above mentioned methods worked. How can i purify or precipitate my desired protein?
Hi there,
Ion exchange chromatography should allow you to concentrate the protein onto the column and to get rid of the contamination.
What do you mean by none "worked"? Did they not precipitate your protein, or did they also precipitate the contaminants? What were the column-based methods and how specifically did they not "work"?
If your protein didn't precipitate, you can try adding carrier, like 500 ug/ml BSA. Acetone precipitation can then target the total protein concentration, no matter which protein is which. So your low concentrated protein would precipitate alongside the BSA. The downside of course is that you'll end up with a lot of BSA in your sample, which may or may not be a problem for your application.
You should used dialysis tube of required KDa's and centrifuge it for long time, otherwise another method if you have used metal affinity chromatography column for purification of enzyme, then try to increase the imidazole concentration.
If you want to get rid rid of the oligosaccharides -and if they are small enough- a simple gel filtration step on a disposable PD-10 desalting column (GE sells those) may do the trick. If you want to also concentrate the sample, then ultrafiltration in a low MWCO Amicon cartridge (these you can get from Millipore) may be your best option, provided there is no non-specific adsorption of the sample to the filter.
Hope it helps!
@ John Schloendorn The contaminants are being precipitated alongside desired protein and interfering with reducing sugar estimation.
You can use amicon centrifugal filter units, these units will retain high molecular weight protein and will eluate low molecular weight contaminants. The size of filter unit vary from 3-100 kda, so you can choose according to molecular weight of protein and contaminants.
Moreover, these filter units can also concentrate your protein.
Best of luck.
Vijay
Hello, you can either try to concentrate your protein, using a centrifugal concentrator (Amicon with the desired MWCO), this will concentrate your sample and allow you to carry on your downstream applications. Otherwise, have you tried to precipitate the protein using the methanol/chloroform extraction?
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