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strep-tag II purification not pure


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#1 hallie55

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Posted 02 January 2011 - 07:57 AM

Hi,

I am having problem getting a pure protein using the Strep-tag II purification protocol. My target protein is Maltose binding protein with Strep-tag II at the N-terminal. The plasmid that I use is pET-51b (+) and was transform into an expression host BL21(DE3)pLysS. The target protein is cytoplasmic and was lyse by Bug-buster and Benzonase. Then, the protein sample was loaded onto a 5ml Strep-trap Hp column. I am using Akta Prime plus machine. And the column i am using is 5ml Sepharose Strep-tactin by GE healthcare. The binding buffer that I use is phosphate buffer and I try to use Tris Buffer too as recommended at different times. Both using phosphate buffer and Tris buffer results show multiple bands at the peak of the elution. Which means that the protein is not pure. But, the more troubling is that the multiple band also shows that there are Strep-tag II. My target protein is ~42kDa. The one that was box in the attach. Is the other bands aggregates? Please advice. Greatly appreciated

SdS page of StreptagII purification of MBP.jpg

Strep-tagII western blot.jpg


Thank you so much

#2 mdfenko

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Posted 03 January 2011 - 08:39 AM

bands below the protein of interest would not be aggregates. they may be fragments.
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#3 hallie55

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Posted 17 January 2011 - 08:33 PM

bands below the protein of interest would not be aggregates. they may be fragments.




Fragments due to? How to get rid of them?

#4 mdfenko

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Posted 19 January 2011 - 08:46 AM

fragments can be due to the presence of proteases or truncation during transcription or translation. or, sometimes, mechanical shearing.

depending on how much difference there is in molecular weight, you may be able to separate them from your protein of interest by gel filtration.

the immunodetection of the bands may also be caused by non-specific binding.
talent does what it can
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