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Purification gone wrong what to do?

Purification gone wrong what to do? - Recombinant Protein Forum

Purification gone wrong what to do? - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


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Old 11-18-2009, 08:27 AM
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Default Purification gone wrong what to do?



Hello there, I'm new to this forum.
I'm currently working on a recombinant protein with a C-term hexa His tag. When I open my cell cultures I got like 5% in the soluble fraction, the rest remain aggregated in the pellet. I run a step of Nickel purification on a column and see 4 bands, 3 bands in addition to my recombinant protein. These additional bands have been identified as various e. coli proteins by mass spectrommetry. I collect fractions and run a step of anion-exchange, again I see the same pattern of contaminant e. coli proteins. Gel filtration indicatates a soluble complex of these proteins as they elute in the same fractions.

I have tried purification under denaturing conditions because I have so much protein in the pellet. The idea is to make purification easy and then refold.

I treat with Urea (8M) in all buffers etc. still I see this same pattern of 4 bands, I try with Guanidine HCl (6M), DTT (10mM), Triton X-100 (0.1%) and still I have these 4 bands.

I simply don't get why I can't separate more efficiently, and have these proteins and be stucked to my recombinant protein.

Any ideas?
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