Avoid Aggregation of protein

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GIBI
GIBI's picture
Avoid Aggregation of protein

Hi,
Could anyone advise me how can I improve or change my purification protocol to avoid aggregation of my protein?
Little Intro:
My protein is about 25KDa (it consists of Thioredoxin, His tags and 60AMK, mean incorporated in pET32B).
I am using Rozetta Cells and cultivate them on temperature 18degree after induction (25degree) of 0.5mM IPTG.
After purification step (chelating sepharose) in PBS Buffer, I leave sample in dialysis overnight and after I do Gel filtration on Superdex 75.
before Superdex I concetrate the sample (there is no precipitation in sample). But on Superdex my protein runs like 200KDa.
On SDS gel it is only my 25kD band.

Please could you help me ? any suggestion?
Thanks a lot
GIBI

Chin Fen Teo
Chin Fen Teo's picture
 Hi GIBI,

 Hi GIBI,

How about adding DTT to prevent disulfide bond formation within your protein during the chromatography?

Cheers.

protoldo
protoldo's picture
Hola, Yes, a bit of reducer

Hola, Yes, a bit of reducer would avoid the formation of -S_S_ intermoleculars, if your protein has them. if you know the isoelectric point, (there are some tools to calculate it, probably in this page or others in internet, knowing the sequence) try to choice a pH of work  distanced of it to improve the solubility, and study induce with less IPTG, 0.1-0.2 mM. If your aggregates are mediated by s-s you could follow the purification without denaturing at all the samples, loading some wells in PAGE with loading buffer but without reducer and withouth boiling. In this way you could see the multimers without waiting to the result of Superdex. Buena suerte

Rajeshwari patel
Rajeshwari patel's picture
 hi,

 hi,
 I am completly agry with the  protoldo and Pippuri apart from that  avoid shaking if possible and reduce the time between two steps.

Rajeshwari Patel