His-tag protein purification

Use of His-tag to coprecipitate a GPCR with protein partners
14.10.2003 10:18
I am working on GPCR-partners interactions, especially on PDZ type interactions.
I detected specific interactions by chromatography affinity using a peptide mimicking the C-ter of my receptor.
But infortunatly, co-immunoprecipitation has failed : It seems that my receptor is not correctly immunoprecipitated.

I intend to realise a coprecipitation beetween my GPCR recombinated with a His-tag on N-ter and protein partners. I would like to investigate this interaction in eucaryotes cells lines likes COS or HEK cells.
Does anyone know a lab staff working on such experiments ?
Do you think that it could success ?

insoluble protein
29.03.2003 23:58
I am working on a small heat shock protein with his-tag.
After I purified my protein in a Ni column my protein is soluble
in the imidazole solution, but when I am trying to dialyze it the protein became insoluble.
SDS-PAGE of proteins
Hassan Darash 18.02.2003 09:54

I run SDS-PAGE for separatoin LMG160 from total proteins of nucleous. But when i stained the gel by coomasiblue I saw that the coresponding band has a stranger shape mean that the flanks of the band migrated toward up. If any one know why this phenoma take place I would appreciated to send it to me. I can send to him/her the picture of the gel
RE:SDS-PAGE of proteins
15.03.2003 09:42
If you are saying what I think you are, that is your band has a "smily" shape, then you may want to make your gel a slightly lower % and also try running your gel a little slower. If you don't use a stacking gel you should probably consider doing that also. Maybe these will help but I am not sure because I don't know what you did and haven't seen the gels.
Problems purifing His-tag proteins
16.12.2002 16:50

I'm trying to purify a his-tag protein expressed in 293 cells and secreted to the culture media. My problem seems to be that serum proteins bind to the Ni resin preventing complete recovery of my protein. Reducing the content of FCS in the culture media drops the yield (expression+secretion). Does anyone know of any good resin with low protein binding properties or a way to get rid of albumin before purification?
RE:Problems purifing His-tag proteins
13.01.2003 22:59
Respected Sir/Madame,
There is a HiTrap Blue HP column from amersham biosciences, also called cibacron blue F3G-A, which is used for removal of albumins. You can try for removal of the serum. After removal of serum you can try from ni resin to purify your protein of interest.
Thanking you,
Kumaran.T
muthen_6@yahoo.com
> I'm trying to purify a his-tag protein expressed in 293
> cells and secreted to the culture media. My problem seems to
> be that serum proteins bind to the Ni resin preventing
> complete recovery of my protein. Reducing the content of FCS
> in the culture media drops the yield (expression+secretion).
> Does anyone know of any good resin with low protein binding
> properties or a way to get rid of albumin before
> purification?
question
05.08.2002 10:33
Who could tell me the sencondary structure of interleukin-1 receptor antagonist?How can I improve the solubility of inclusion body of IL-1ra.Many thx for ur considering.If U know the knowledge plz contact me at 0086 23 62813515 or thd882000@yahoo.com.

2002-07-22 00:48:15
Theodore (thd882000@yahoo.com)
good vector system
05.08.2002 10:32

Can u suggest me some good vector system which will give small his tag(6residues) at N-terminus or c-terminus immediate to the gene?
The vector should have promoter other than phage promoters (i.e they should not have any requirement for specific phage RNA pol) as gene which i want to clone is toxin for e.coli and can be expressed only in resistant stains.

2002-06-20 01:46:05
Kanika Bajaj (kanika@mbu.iisc.ernet.in)
RE:good vector system
22.10.2003 08:35
>
> Can u suggest me some good vector system which will give
> small his tag(6residues) at N-terminus or c-terminus
> immediate to the gene?
> The vector should have promoter other than phage promoters
> (i.e they should not have any requirement for specific phage
> RNA pol) as gene which i want to clone is toxin for e.coli
> and can be expressed only in resistant stains.
>
> 2002-06-20 01:46:05
> Kanika Bajaj (kanika@mbu.iisc.ernet.in)

i have a vector derived of pRSET which have 6xhis near to the AUG (between 4 or 5 aa) if you are interesting in this please contact me at rtapia@ins.gob.pe
RE:good vector system
22.10.2003 08:20
>
> Can u suggest me some good vector system which will give
> small his tag(6residues) at N-terminus or c-terminus
> immediate to the gene?
> The vector should have promoter other than phage promoters
> (i.e they should not have any requirement for specific phage
> RNA pol) as gene which i want to clone is toxin for e.coli
> and can be expressed only in resistant stains.
>
> 2002-06-20 01:46:05
> Kanika Bajaj (kanika@mbu.iisc.ernet.in)

Hello I hope that you need help yet. I have a vector derived of pRSET (invitrogen) which have 6histag near to the AUG (4 or 5 aa) in the N terminal it have the T7promoter . If you are interesting in this please contact me at rtapia@ins.gob.pe
comments
05.08.2002 10:31
Many comments on your problem:
First
for solubilizing your protein you can use Guanidium hydrochloride 6M it's the harshest solubilizing agent.
second
Solubility is probably the most difficult issue, lower temperature for induction time and lowering the expression is usually helping for soluble production.
Third
Of course denaturation can be a problem there are some refolding protocol available both after purification or more recently directly on the column by washing the bound protein with a gradient wash of decreasing concentrations of denaturing agent or by using oxidized glutathine in saline buffer. After refolding the protein can then be eluted with imidazole gradient(in the case of His tag)

Hope that might help you. there are of course tons of others possibilities like co-expression of chaperones molecules to help proper folding and therefore solubility but unfortunately it vary from protein to protein and nobody can give real prediction
So jut try out as much as you can.
Feel fre to e-mail if you have specific questions
Pesji


2002-06-18 06:08:41
pesji (pesji@yahoo.com)

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