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His tag protein purification problem - (Jun/12/2004 )

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InvisibleSurfer:
I want to express a enzyme in E.coli, how can i get rid of the DNA !Can you give me the protocol!
thanks

-David Zhang-

QUOTE (David Zhang @ Jun 15 2005, 05:42 AM)
InvisibleSurfer:
I want to express a enzyme in E.coli, how can i get rid of the DNA !Can you give me the protocol!
thanks


I am isolating a DNA binding protein expressed in E.coli, and I noticed that a large amount of host DNA is bound to it.

I am going to use an additional precipiation step with Polymin B or PEI 10%.

-dtle-

dle:
thanks a lot!
Can you give me the detailed protocol or referrence!
How can i get rid of the redundant PEI after precipiation step with PEI?

-David Zhang-

QUOTE (David Zhang @ Jun 17 2005, 12:33 AM)
dle:
thanks a lot!
Can you give me the detailed protocol or referrence!
How can i get rid of the redundant PEI after precipiation step with PEI?


I am using 10% polymin B to precipitate the protein (~350ul polymin B for 10mL of extract), keep on ice for 20 min, then centrifuge at 10000rpm to collect the precipitant. Then dissolve the precipitant by TGED buffer with 1M NaCl, pipetting to break the pellet. Keep on ice another 20 min and centrifuge at 16.000 rpm, collect the liquous. At this time use Amonium sulphate to precipitate the protein again, keep on ice for 20min, then centrifuge.

Collect the pellet and dissolve it in the lysis buffer (or binding buffer).

Goodluck, I am trying and still did not check the outcome yet!

-dtle-

Hi everybody!

I would be soo glad if anyone could help me. My purification of his-tagged scFv drives me crazy...

The problem is that the protein which is definitely expressed (western analysis) disappears after binding to the column. I cannot detect the protein in any fraction (flow, wash, elution, even after stripping of column!)...

It is a 34 kDa protein, expressed in a pET-based system, extracted from periplasm fraction.
First, I tried a Superflow His- column (Amersham) with PBS-buffers (with correct pH). I used 0.5 M NaCl and a imidazole-gradient up to 500 mM. I got some Peaks during the run, but in western blot analysis no target-protein was detectable, although Ponceau-Staining showed many bands in these fractions.
Then I used EDTA-free protease inhibitors, with the same effect.
Next step was to try overnight-binding to the column. But then, I got no Peaks...
I tried an other buffer, a Tris-buffer, because Tris 'may' reduce binding. No Peaks.
I changed column, used a "normal" His HiTrap Column (Amersham) with direct injecting and overnight binding. Also no Peaks.

Now, I'm going to purify a control-scFv, but perhaps you have some suggestions...

One thing at least: I tested this protein also in ELISA (direct coating and binding to antigen) and got good positive result, so the His-tag shouldn't be internalised or something like that...

Thank you for your replies!

-Janina-

QUOTE (somali @ Feb 12 2005, 04:23 AM)
Hi All
        I have very less experience with His tagged protein purification. After purifying my protein (30kDa) using Ni2+ resin I still see other proteins in the eluted fraction. One of my colegue demands that by re-purifying those fractions in the Ni2+ resin once again under exactly the same conditions he can clean it up . Is that possible?
Thanks for your suggestions,
Somali


Hi
i also jave a similar problem. try by increasing the concentration of imidazole in the wash buffer and elution buffer.

-sudarshana_p-

hi all

I am having problem with purification of my His-tagged protein (105 KD). after elution many other proteins are also getting eluted. some of u have suggested to use a concentration gradient of imidazole. what should be the gradient from lower to higher or reverse? At present I am using 10m M Imidazole for binding, 20mM for wash and 250mM for elusion. I am keeping the pH of the buffer at 7.9

also can you enlighten me about Nacl concentration gradient? would that help? if yes then what should be the concentration gradient like?

thanks
sudarshana

-sudarshana_p-

Hi there,

I am working with an extremely hydrophobic protein, Vpu, 24 KDa in length including the poly-His-Tag. I have cleaved the His-Tag using cyanogen bromide and am endevouring to separate the 2 fractions using the Ni-NTA beads. The problem is that the protein sticks to the beads more than the His does! I have used a pH gradient from 6.3 to 4.5 to no avail... all the His is gone but the Vpu is still there. My next step is to try an imidazole gradient... any tips on what concentrations and any other ideas??

unsure.gif

lala

-Llara-

imidazole gradient: i use it to purify an integrase protein (32 kda) which has a 19-mer his tag on it. 10, 60, 150, 300, 450, 600, 700, and 1 M imidazole gradient works for me... but make sure your gradient is very steep, i.e smaller quantities at each concentration, with the smallest at the fraction you think its going to come out in. for 10, 60 and 150 i use 2 ml, 300 1 ml, 450 1 ml, and 2 ml of 600, 700. 3 ml of 1 M. good luck!

-radgrad2008-

Hi all, aren't all proteins suspiciously highly unstable and easily 'lost'. How to make sure your protein is still in working 'condition'?

-paperclip-

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