Protein in each elution step during His-Tag purification - (Jul/23/2007 )
Hi everybody,
I've got a problem in my His-Tag Purification.
I'm overproducing a 58 kDa Protein in E.coli. Most of the Protein is unsoluble (I tried several growth temperatures and IPTG concentrations) however I put the soluble fraction on a Ni-NTA-Agarose Column (Quiagen). I detect my Protein by Anti-His-Antibody in each of the elution steps. The most is the flow through and in the washing step but also some in the fractions of 50,100,150 and 200 mM Imidazol. I don't think that the column is saturated.
My buffer is 50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazol pH7,5 or pH 8
Should I try higher amounts of NaCl?
Has anybody an idea?
Thanx a lot
I've got a problem in my His-Tag Purification.
I'm overproducing a 58 kDa Protein in E.coli. Most of the Protein is unsoluble (I tried several growth temperatures and IPTG concentrations) however I put the soluble fraction on a Ni-NTA-Agarose Column (Quiagen). I detect my Protein by Anti-His-Antibody in each of the elution steps. The most is the flow through and in the washing step but also some in the fractions of 50,100,150 and 200 mM Imidazol. I don't think that the column is saturated.
My buffer is 50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazol pH7,5 or pH 8
Should I try higher amounts of NaCl?
Has anybody an idea?
Thanx a lot
I think salt concentration is not a cause of your problem! Mainly salt is used to decrease non specific matrix binding of other protein impurities in your sample.
What is about lengt hof His-tail? If too short, so may be weak binding with NiNTA matrix? Did you use EDTA in previous steps of cell lysis? If so is possible leakage of Ni from column, also did you use BME or DTT at concentrations more that 20mM?
Concerning unsoluble protein - may be your protein in inclusion bodies? So follow strategy for IB purification!
Thank you for the quick answer.
The His-Tag consists of six Histidines. I did't use any EDTA, DTT or BME...
If no protein would bind to resign, I could understand but some does so...
Do you think longer Incubation of agarose with the protein could be an idea?
I also thought about purifying from IB but my protein containes Co-Faktors. I'm going to loose them (assuming that they are there) upon the purification from IB, right?
Thanx so much!!!
I've got a problem in my His-Tag Purification.
I'm overproducing a 58 kDa Protein in E.coli. Most of the Protein is unsoluble (I tried several growth temperatures and IPTG concentrations) however I put the soluble fraction on a Ni-NTA-Agarose Column (Quiagen). I detect my Protein by Anti-His-Antibody in each of the elution steps. The most is the flow through and in the washing step but also some in the fractions of 50,100,150 and 200 mM Imidazol. I don't think that the column is saturated.
My buffer is 50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazol pH7,5 or pH 8
Should I try higher amounts of NaCl?
Has anybody an idea?
Thanx a lot
as I use Tris as buffer component, I am wondering if the phosphate disturbs by complexing with Ni2+; check with free ion calculation program (f.i Eqcal) or change the buffer
I think that longer incubation will not help you! About co-factors - What kind of cofactor is for you protein , if low molecular like nucleotides or metals I think you may add these cofactors into refolding buffer for proper refolding but if cofactors are more sophisticated it will be a serious problem to get properly folded active protein after renaturation during isolation from IB . So may be you should correct your expression system to enhance cytoplasmatic production of your protein Another way is change strategy for purification. First try IEC ( better if you know pI ).