Hi, I try to purify protein expressed HEK293T
I've tried many things, but I failed...
* Binding: 0 mM Imidazole, Wash: 20 mM Imidazole, Elution: 500 mM imidazole
* All buffer contain protease inhibitor cocktail without EDTA
1. Increase NaCl concentration (150mM, 300 mM, 500mM)
[50mM Sodium phosphate buffer (pH 7.4), 150~500 mM NaCl]
I thought the low stability of protein causes aggregation
When I use buffer including 300mM NaCl, the intensity of target protein band is getting stronger than 150mM in CBB staining after elution and centrifuge (to discard precipitaion)
In 500 mM, there are too many non-specific bands..
2. Change the buffer
[50mM Sodium phosphate buffer (pH 7.4), 150~500 mM NaCl]
I used 50 mM sodium phosphate buffer (pH 7.4) and I change to 50 mM Tris buffer (pH7.4)
There was no change.
3. Add the TCEP and 10 % Glycerol
[50mM Tris-HCl (pH 7.4), 300 mM NaCl, 1mM TCEP, 10% Glycerol]
It was a little helpful, but the problem is still not solved.
4. pH gradient elution (binding pH 8, Wash, pH 6.4, Elution: pH 5)
It doesn’t work. I mean, after elution, The protein was still binding to the bead.
The pi value of our target protein is approximately 4.9.
Our protein pi value is similar pH range of the elution buffer, so I think protein is aggregated in the column. Also, there are a lot of non-specific bands
[-NEXT TRY-]
pH 8 Tris buffer, 300 mM NaCl, 1 mM TCEP, 10% glycerol, 0.05% Tween 20
Imidazole (Binding: 20 mM, Wash 50 mM, Elution 500 mM)
- I checked that target don't elute in 50 mM imidazole
- I said, there are too many non-specific bands.
I heard that the little basic buffer and detergent help to inhibit precipitation
Can I use this buffer? Can the problem be solved?
Is there any other solution? Please make a suggestion...
I can't use other tag or remove tag.. I want to solve this problem by changing the buffer composition.
Thanks in advance.
Dear Sunha Hwang
Is it you protein a soluble protein and is it expressed in the HEK293T surnatant?
Because iin this case t is quite strange that it is stable for some days in solution at 37°C and it tend to precipitate and/aggregate after the IMAC elution.
Is it your eluted fraction coloured? Is it possible that you protein has free cisteines or many histidines and it can extract some nickel from the coloumn and the metal drive the aggregation?
Did you tried to add some EDTA immediatelly after elution?
Alternativelly it seems that your protein has low stability under concentration. In this case you can try to dilute it immediatelly after elution and see it the precipitation happen also when it is stored at low concentration.
In this case is possible that addittion of some additives (eg arginine, sucrose, weak detergentes as chaps) may improve the protein solubility but is not sure, since it depends a lot if this aggegation propensity is due yo hydrofobic or charge interactions.
As first trial, i suggest to you to reduce the imidazole in elution eg 300mM (pH 8 is ok) and add 10mM EDTA in the eluted fraction just immediatelly after elution and see it this change.
if you do not see any improvment, you have to check well you protein sequence to see if there are some charge or hydrophobic patches that may induce aggregation under concentration,. but it is a more complex work since theoretically to do it you need at least a 3D structural model of it.
good luck
Manuele
Manuele Martinelli I would like to add another point to your answer. Since pI of the protein of interest is 4.9, a buffer pH of 7.4 seems so high. Ideally, the pH of buffer should be at least 1 pH point lower or higher than pI and should be closer to neutral as possible. So, I would suggest using a buffer in pH of around 6 instead of 7.4.
Some comments:
IMAC is not specific enough for you to not see other proteins being adsorbed.
IMAC must be run with at least 500 mM NaCl to reduce electrostatic interactions.
IMAC must be run at least at pH 7 to have an unprotonated histidyl residue.
As Manuele Martinelli comments, you need to establish where the protein is. EDTA washing must be run, if what comes out is turbid, your protein is aggregating.
Manuele Martinelli, Hi. I checked this protein is soluble via Western blot.
- I saw some particles (turbid) in eluted fractions right after elution...
- this protein has an S-S bond and I'm not sure some residues cause extract Ni ions... I will purify using the above buffer conditions and then check the change by adding 10 mM EDTA as you suggested.
- It is right to add the EDTA in eluted fraction right after elution? not bead?
- You mean, the final concentration of EDTA is 10 mM?
Tamilselvan Dhakshinamurthy
I thought that you said, but I found it in qiagen handbook:
The histidine residues in the 6xHis tag have a pKa of approximately 6.0 and will become protonated if the pH is reduced (pH 4.5–5.3).Under these conditions, the 6xHis-tagged protein can no longer bind to the nickel ions and will dissociate from the resin.
Omar Gonzalez-Ortega
I'll increase NaCl concentration to 500 mM and add the EDTA
Thanks everyone, I hope I hope this problem is solved
Dear Sunha Hwang
yes you can simply add 10mM final of EDTA just after elution. EDTA is bad only in the case that your protein is a metallo protein that need to bind the metal for his function, since EDTA will extract it. but not in case that the metal binding is not specific, has may happen in the case that the metal is stripped from the coloumn.
good luck
Manuele
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