Hi David

Are you loading too much protein into the column? that causing aggregation anyway sequence both proteins, once you identify which one is your target protein (could be both?) then stick with the peak & fraction corresponds to it, after which you may need to use a different chromatography step to get it purer. One more thing is it not dimmer? what size are looking for?

Hope this helps   

I am not loading too much protein. Both are the target protein ( I would think that 11,3 ml peak is a hexamer and the 9,8ml peak is a double hexamer). I did these chromatography steps: HisTrap, MonoQ and Superose 6. The protein was almost pure ( I had a little contamination, which gene is Arna, but I couldn't separated it of my protein).

We dont know the protein structure but with this results We expected the protein form hexamers or heptamers and this one could bind in a double hexamers or heptamers.

Thank you

That chromatogram at page  5 reminds me of my own chromatography :o))

Do you need to perform the chromatography at all? It seems rather pure (if we talk about the first gel in your Word).

Dear David:

Which kind of protein are you working with?

You have to be careful with the additives you used when working with multimeric proteins because the additives can interfere stabilising/disrupting interactions between the units, etc. General conditions to perform purification should be similar to those the protein has within the cell (pH, temperature, ionic strengh, etc.). For redox proteins, the use of reducing agents in the buffers helps....the use of high salt concentrations improves halophilic proteins stabilisation...

I suggest you to look for works were this protein or similar proteins have been previously isolated as well as works about the physiology and biochemistro of the organisms from where you can isolated the native protein (these will give you information about the optimal physiological conditions for your protein...try to mimetic them).

Hi Rosa, First thanks for your answer.

The problem with my protein is if you don't add additives like glycerol It forms aggregates easyly and this is a problem. We suggest that the peak which you see in Ve 10,5ml is a unfolding structure protein and when We add additives the protein folds in the native structure ( It's suggest the 11,3ml peak) and It could form the double hexamer ( 9,8 ml peak).

We will expect that. This week We are going to do DLS

Stability protein in the purification. Could you help me? - ResearchGate. Available from: https://www.researchgate.net/post/Stability_protein_in_the_purification_Could_you_help_me#57c7e3b8b0366dc371176e27 [accessed Sep 1, 2016].

So you have confirmed in both peaks is your protein, right? How about to try circular dichroism to make sure it's not denatured?

In case it's native, I think you just showed that your proteins form hexamer and dimer of hexamers. Pretty cool, isn't it?

Is your protein a membrane protein or a protein interacting somehow with the membrane? Is it native or a recombinant protein over-expressed in vectors like E. coli?

Another thing that occured to me on your other question - did you try to centrifuge your sample for a long time? If it is aggregated, it will form pellet. If it's native (even as multimer), it should stay in solution. So this could be the fastes and simplest way to tell.

Yes Thomas. The protein is in both peaks ( We sequenced its).

Hi Rosa. We don't know so much information of our protein but we think It isn't a membrane protein. It is inside of the bacteria with a structural function. It's a recombinant protein.

Thank you.

 I used Google Translate to read your attached notes (which translated most but not all of the words, so I might have misunderstood some of your notes). From what I understood, the sizes that you are getting from the gel filtration chromatography peaks are multiples of the recombinant protein that you cloned and expressed. The larger sizes could be due to association of the recombinant polypeptide chain, or contaminating proteins that bound to the cloned and expressed protein. Did you consider the possibility, if you have any cysteine residues in the recombinant protein, that mis-folding could expose the Cys residues so that disulfide bonds formed between polypeptide chains. Mass spectrometry can help determine if there is contaminating protein or degraded recombinant protein in the gel filtration peaks. To determine whether the association of subunits is due to misfolding or a characteristic of the native protein, I would think that you would need to study the protein from the natural source, not the recombinant protein. 

It's very interesting your answer but We dont think that the contamination could bind with my protein because It is in all peaks ( 9,8-10,5 and 11,3 ml peaks) in the same concentration. We expect the 11,3 ml peak is the hexamer and the 9,8 is two hexamer binding ( Next week I am going to do DLS)