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protein purification - Protein Forum

protein purification - Protein Forum


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  #1  
Old 08-19-2010, 03:18 PM
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Default protein purification



all,

I'm working on a low molecular weight protein with antimicrobial activity.
Initial purification was by precipitation and passing throu gel permeation sephadex G 25 column(elution 1-5kDa) and got activity at a particular fraction.
In order to find out the molecular wt. tricine sds page was done with the active fraction.
And to my surprise instead of a single band i got two bands one nearly at 15 kDa and other at 4kDa. please suggest me solutions to this to proceed my work.
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Old 08-21-2010, 01:18 AM
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Default Re: protein purification

Hello,

Well, it it difficult to tell without more details, especially about the precipitation step used in purification. Perhaps you prefer not to reveal too much?

Three possibilities suggest themselves.

1. The protein is not pure, but contains a contaminant at 5 kDa (this will not be removed by gel-filtration on Sephadex G25, which has an exclusion limit of about 5 kDa).

2. The protein is a heterodimer with subunits of 5 kDa and 15 kDa.

3. You have got proteolysis during purification. Does the purified protein still have anti-microbial activity?


What happens if you gel-filter on a long column (1 m or so) of Sephadex G50 (or Sephadex G75) ? Do you get a single peak, and what is the apparent molecular weight? That is, do you have any idea of the molecular mass of the native protein? It may not be a monomer in solution! It could be tetrameric, for example, with a native Mr of 60 kDa.

Good luck,

tgd
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Old 08-23-2010, 01:45 PM
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Default Re: protein purification

hi,
thank you for ur suggestion.
the protein still has activity after gel permeation which eluted.
i'l try out with G50 which has a wider range of seperation.
Or planning for a native page

thanks,
sarath
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Old 09-20-2010, 03:48 AM
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Default Re: protein purification

I agree in the sense that you may just be seeing degradation with may be just happening on your gel. I am sorry that I can not think of the paper but look up VCATH protease and E-64. I have added E-64 to my 5X SDS because of this bugger of a protease
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