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Protein Aggregation with 6xHis tagged CreGFP


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#1 Aallea

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Posted 14 July 2011 - 12:35 PM

Hi, I've been purifying Cre fused to GFP with a 6xHis tag by running it through an NiNTA column. At first I was pretty excited about it because we got a great yield (7.8 mg/ml), and you could really tell because the whole thing glows bright green in sunlight. However, it seems to form aggregates really quickly, and in huge amounts. I eluted the protein in 50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, 3 mM PMSF, pH 8.0. It started to form aggregates overnight. Then I did a dialysis with PBS. The next night a huge portion of the protein had formed a solid aggregate.

Does anyone have any suggestions for how I could prevent this aggregation, or even possibly undo it?

#2 protolder

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Posted 14 July 2011 - 10:28 PM

Hi, I've been purifying Cre fused to GFP with a 6xHis tag by running it through an NiNTA column. At first I was pretty excited about it because we got a great yield (7.8 mg/ml), and you could really tell because the whole thing glows bright green in sunlight. However, it seems to form aggregates really quickly, and in huge amounts. I eluted the protein in 50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, 3 mM PMSF, pH 8.0. It started to form aggregates overnight. Then I did a dialysis with PBS. The next night a huge portion of the protein had formed a solid aggregate.

Does anyone have any suggestions for how I could prevent this aggregation, or even possibly undo it?

Hola if you know the isoelectric try working a bit far away of it where the solubility is more high, if no introduce the sequence of your protein in any of the tools for calculating isoelectric point.Congratulations by the high level expression, you have had your protein soluble and pptation has coming by the high concentration and could occurs that you have overpassed the maximun of solubility. you can add some glycerol 10-20% and manuals suggest phosphate better than tris in the buffer but I donīt think that this affect in a big part in solubilization. Buena suerte again




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