Dear Ha

Just some question that may suggest to you a possible reason:

1) Is your proteins contain cysteines?

Because is it possible that with the exposition at the air and at high concentration that you have in the elution, if in your sample there are free cysteines is it possible that there is aspecific intra-molecular S-S bond formation which induce protein aggregation and precipitation

To check it, you can load the precipitate in SDS page in presence and absence of DTT (non-reducing SDSpage).

Some example of non reducing SDS-page are reported in the ProteoCool n°15 on my blog ProteoCool (https://proteocool.blogspot.com/p/page3-protein-puriifcation-and.html)

In this case the addiction of DTT in your buffer just after elution may protect your sample from precipitation. However, it depends if the cysteines have to form S-S bonds because in this case the addiction of DTT will maintain your protein soluble, but it will be unfolded.

2) Is your protein contain a conserved metal binding motif? (eg HXXH)

Because in this case is it possible that the protein extract Nickel from the column and it can induce intra-molecular aggregation. In this case the addiction of EDTA just after elution my protect from aggregation.

3) Which is the pI of your protein and the pH of your elution buffer? Because in the pI is too close to the pH is it possible that the protein is not charged and poorly soluble in water buffer.

Ciao

Good luck

Manuele

Did you by any chance add the 1 mL of NiNTA directly from the storage bottle? Be aware that resins often are stored in 20% ethanol and need to be washed/equilibrated before use. If you carry over ethanol into your lysate you'll surely observe precipitation...

Hi Sebastian Schmitt

Hi Manuele Martinelli, thank you for your help. Here are my answers to your questions:

1. My protein has three cysteines. Two are at the N and C and formed a disulfide bond. My labmate suspected that adding DTT might break the disulfide bond and unfold the protein, leading to aggregation. I had tried with and without DTT but the precipitation was still there.

2. This is a good suggestion to look into. How do you check for the metal-binding motif? I tried online software but my protein was too small for the software to tell.

3. My protein pI is 8.45. I use lysis buffer pH 7.4.

Dear Ha Pham

Generally i'm performing BLAST using the protein as imput and i'm checking in there are conserved Histidines, aspartates, glutammates or other residues that are suggested to bind metal in literature.

I'm don't know if there is a reilable software able to identify the metal binding from primary sequence. You can try SMART to EMBL (http://smart.embl-heidelberg.de/) which is able to identify conserved domains but i'm nor sure that it work so well with metalloprotein.

just 2 more questions:

1) why are you using the tritonX100 in the lysis buffer? Did you remove it in the elution?

2) IS your protein expressed in E.coli? And in this case, are you using Origami strain or periplamic expression to help the S-S formation?

ciao

MAnuele

Hi Manuele Martinelli ,

1. I used tritonX100 with the hope to solubilize the protein. I have tried lysis buffer without TritonX100 and with 10% glycerol to stabilize the protein. However, the result was the same.

2. I used E. coli BL21 RIL codon plus (Stratagene) for expressing.

Thank you,

Ha

Dear Ha

i think that if to solubilize the addiction of TritonX is required, probably the protein is not correctly folded and i suspect that it is due to the incomplete formation of S-S bonds which cannot happen in cytoplasm in the BL21 cells and that the precipitation happen once you remove the TrytonX.

I suggest to you to try to express the protein in the Origami strains (or Rosetta Gami if your need the codon supplementation) and see if you can obtain some soluble form with out the addiction of the Tritonx.

Ciao

Manuele