Hi Madison, does your protein have free sulfhydryls? That could explain why your protein aggregates after affinity purification.

If so, you can add 1 mM glutathione to your protein solution for 1 Hr. at RT and dialyze it against the Ni++ column buffer. Then put it through the Ni++ column, elute and see if it precipitates.

I had good results using the method proposed by Golovanov:

https://www.ncbi.nlm.nih.gov/m/pubmed/15264823/

With Nickel-affinity, you should keep in mind that leaking Nickel ions may lead to unwanted elution of his-tagged protein.

Also, leaking Nickel ions may form a white precipitate with imidazole, that might sometimes be mistaken for crashed out protein. Co-precipitates imidazole-protein are also possible.

HI Madison, its a bit hard to answer the question without knowing what kind of protein you are trying to express and purify. However, from the sound of it, it seems you are trying to express proteins in E coli or Sf6 cells, which are not the protein's native environment. Therefore, you probably do not have (a) either the proper chaperones or (b) interactors that probably stabilizes the protein and ensure solubility.

With that said, have you tried using a different purification strategy? Maybe GST or MBP? Maybe these larger, well folded proteins could help with the solubility of your protein better, and if your insert a cleavage site, you can remove them afterwards if needed.

I have tried with an MBP tag in e. Coli and this helps with expression and solubility. I cannot remove the tag in e. Coli otherwise I lose most if not all the protein to aggregation. Your a and b ideas are very likely correct, but I don’t know what chaperones it has. It binds RNA.

I use B-me as a reducing agent, this should work the same as glutathione right?

Hi Madison, thats a difficult situation. BME or DTT will interfere with your HIs-Tag purification. you could try MBP or GST, but when you cleave the tag off maybe keep a known amount of BSA in your solution as a stabilizer if possible.

The concentrations of b-me or DTT I use are ok according to the manual for the ni-NTA beads.

Yes I have tried both 300 mM KCl and 500 mM nail. I have also tried using L-arginine and L-glutamate.

Peter, thank you for the paper, I have read this paper and tested out the 50 mM of Arg and Glu concentrations without success.

The precipitate should be protein as I have recorded concentrations using bradford throughout the purification and can see the concentration of protein decreasing, even as I am trying to concentrate the solution. The imidazole protein precipitates are an interesting idea, although I have noticed aggregation that occurs upon removal of imidazole through dialysis. For this reason I have also tried adding EDTA to protein fractions right after elution from nickel column.

Dear Madison,

As mentioned before, you do not say much about the protein. Without it, it is difficult to help you.

Based on the discussion, I believe it looks like you are having 2 effects in the protein: a) high charge (RNA binding?) and b) disordered regions. Conventional systems will not work and I suggest on-column refolding.

Prepare inclusion bodies of your 6xhis-tagged protein in 6 M urea and 10 mM imidazole, load them onto a gravity column using manual loading or a peristaltic pump, wash off the crap and then try 1-2 hr refolding using a gradient of urea and final buffer (1xTBS). After washing off contaminants with 20-40 mM imidazole, try releasing the protein using high salt and 100mM or 300mM imidazole. Salt should shield your charge and prevent aggregation. 40-50 mM Arg could be added as well.

BME or DTT can be easily taken care of if you remove unbound Ni ions using GE recommendations. The Ni will stay green on the column up to 2 hrs in low concentration DTT or even 30 mM BME. If you think there are free SH groups, use 1-2 mM TCEP (neutralized) in your buffer. After elution, add EDTA and dialyze against 1-2 mM TCEP and EDTA.

GSH is used for refolding if you have disulfide bridges. It can help forming them and typically up to 2 bridges will work fine. More requires a different system.

Dear Madison,

Depending on your experiments, you may also include a mild detergent. Set up a screen using Ni-ag tagged plates (variation from Sigma) and try different conditions to remove it. You may add GFP as a fusion to your protein to follow fluorescence upon removal from plate.

Best regards,

Wes

Is your protein aggregating, or is it phase separating?

Do you know if a lot of protein stays on the column after elution (is it i.e. GFP labeled?)?

If your protein is phase separating during purification, increasing salt in your binding buffer or increasing working temperature (work at RT) might keep your protein soluble.