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Helppurification of expressed protein

protein purification E. coli expression amm.sulf. precipitation HIC p-sepharose

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#1 Always_student

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Posted 10 March 2013 - 09:58 PM

Hi
I have expressed my protein in E. coli. and I want to purify it. I performed the ammonium sulfate precipitation with 15%, 25%....75% in the indicial samples and run both the supernatant and precipitates on SDS page i noticed that my protein starts to precipitate even at 15% (at this conc half of the protein was in pellet and half was in supernatant). at 35% all the recombinant protein was precipitated and was present in gel. Now i want to do purification of that protein by Hydrophobic interaction chromatography using P-sepharose. I am wondering
1. what should be the concentration of ammonium sulfate in the sample ? (normally it is recommended to be 1 to 1.5 M but at this my protein precipitates).
2. what should be the conc of ammounium sulfate for column equilibration
3. What is the appropriate ammonium sulfate conc for wash buffer and for elusion buffer?

#2 mdfenko

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Posted 11 March 2013 - 06:05 AM

1) you can determine the optimum loading concentration of ammonium sulfate by trying a range of concentrations from 0.05M to 1.5M.

see this excerpt from the hydrophobic interaction handbook:

Screening for binding (salt) conditions
1. Using the selected medium and buffer from the previous protocol, set up a series of start
buffers at the same pH, but with reduced concentrations of ammonium sulfate in each buffer
(e.g., from 1.5 M, 1.0 M, 0.5 M with a lowest concentration of 0.05 M).
2. Repeat steps 2–7 from the previous protocol for each salt concentration.
3. Determine the salt concentration that permits binding of the target protein(s) while contaminants
either wash through or remain bound to the column. Determine the lowest salt concentration
required to achieve complete elution of the target protein.

(you can download the handbook from this webpage)

2) the column should be equilibrated with the concentration you determine for your sample.

3) wash with the equilibration buffer. elute with a gradient to 0M ammonium sulfate or with a step to 0M.
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#3 Always_student

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Posted 15 March 2013 - 02:03 PM

Thank you.





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