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his tag protein purification

his tag protein purification - Protein Forum

his tag protein purification - Protein Forum

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Old 10-22-2003, 02:52 AM
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Default his tag protein purification

Hi, my name is Humberto. I'm am currently working with the TOPO TA cloning kit, which I used to obtain my recombinant protein. Using the vector pBAD TOPO, the recominant protein I get is of about 13 kD taking into account the V5 epitope and tandem section of histidines. I performed all the pilot _expression essays to determine the time and concentration of arabinose to best induce the production of my protein from this vector. Once I did this, I grew a 50 ml culture to use in the purification of my protein using the probond system procedure.

Before I started to purifie it, i resuspended the pellet of cells in native binding buffer from the probond system( because I want to preserve the activity of the protein) sonicated the cells and follow all the native purification steps...so I obtained the supernatant and performed a SDS-PAGE and used the comassie blue staining to visualize the protein. I was able to see the protein, loading 10 ul from the 8 ml volume of the supernatant. The intensity of the band was high. Then I prepare the column with the probond resin and once ready I loaded the lysate just like the protocols mentions, and did all the steps. In the native binding buffer I didnt use imidazole. just in the wash and elution buffers. I collected the wash supernatants and the 1 ml elution fractions and did another sds page. I loaded 12 fractions and a sample of the washes collected. The results I got were that I could not see the protein in the elution fractions except in the elution fraction number 1 in which I barely see it with a bit of imagination, but not in the other 11. I also see a few high molecular proteins in the first 3 elution fractions. But I think that my protein is not binding the resin because in the first supernatant which is the one that I obtain after the binding step, I see my protein with the same intensity as the first sds page I ran. And in the other washes i see the protein too, with less intensity though.

I checked the troubleshooting section of the probond manual, but one of the answers I can come up with is that the folding of my protein may influence the proper binding with the histidines.

I checked the solutions. pH and everything. Do you think that the concentration of my protein in the lysate could be a reason or the concentration does not influence in any way the binding?

I dont want to try the denaturing procedure of the probond system because I want to preserve the activity of the protein. Does any one have any suggestions to solve this problem?

I'd really appreciate it.

thanks in advance,

Humberto O.

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Old 10-23-2003, 10:51 AM
Duncan Clark
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Default his tag protein purification

Historians believe that in newspost
<[Only registered users see links. ]> on Wed, 22 Oct 2003,
[Only registered users see links. ] penned the following literary masterpiece:

If the His's are folded within your protein then they are not available
for the column to actually 'see' to bind to.

Assuming the above isn't a problem then what happens if you dilute your
sample say 5 fold and try loading that?

It may simply be that the initial protein 'soup' is really too high a
concentration, plus the presence of nucleic acids in it, and is stopping


I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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Old 11-30-2003, 04:51 PM
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Default his tag protein purification

I thought the premise was that 'masking' was effected, in this case, by
folding of the protein within itself and not related to the concentration of
molecules sorrounding it. Well....

Rolands G. Aravindan
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Duncan Clark wrote in message <6xMlqADaK7l$EAoQ@[]>...

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