Just started to test at what conditions this sample is completely dissolving. I realized that after resuspension when there are still some crystals visible, the sample turns completely white when cooled (I am working on ice). This is reversible. When heated up at 37C for 20 sec it turns completely transparent. This can be repeated.

In the condition where there are no crystals visible, this phenomenon is not observable.

I don't know if this insight helps, but I thought I share it.

The only substances in your description, other than the protein, that are solids and present at some point in large quantity are guanidine and KCl. Since you loaded more sample onto the column than usual, my guess is that the removal of guanidine and KCl were not complete, especially since the protein eluted early, when the salts elute. Since these are not volatile substances, they would have carried through the entire procedure. Both are soluble in water, but not infinitely, so when you dissolve the product in a small volume, the concentrations of guanidine and HCl are near saturation, and above saturation when the solution is cooled.

If you are going to scale up the procedure, you should also scale up the column. If that is not feasible because of cost, then just run the low-scale chromatography multiple times using the low-scale method.

Thank you very much for the reply Adam B Shapiro ! Was hoping you would answer since you are quite an expert in protein purification and biochemistry ;-)

The gravity flow SDBL columns from Phenomenex are the largest there are. So, there is no chance that I can scale up in that matter. I'm also quite surprised that this happens when I load protein corresponding to about "just" 3% of the column since previous RPC runs were actually quite low scaled (about 1.5% weight of Colum protein loaded)

Anyway, what I would take away from this is that if I have the same setup again (more protein to run over the RPC) then I would go back and load as much as I did in previous batches since there might be a problem with salt and guanidine sticking on the column for some reason.

If I would do the same thing again would you think that this issue can be solved by washing the column with more wash buffer (5% ACN) after loading to get rid of excessive salt and guanidine?

And would you think that I can dissolve my protein in the binding buffer again (2M GndHCl, 5% ACN, 0.08% TFA, 200mM KCl) and just perform another RPC and lyophilize again? According to the SDS-PAGE there is not much protein that I lose in the flow-through and none in the wash. Also, during lyophilization, I should not lose any protein. The only thing I could imagine is that the quality of the protein worsens as you can see in the SDS-PAGE where some degradation product appears after elution.

Thanks again and I'm looking forward to your response.

If the protein binds to the column under loading conditions, then you should be able to wash out the salts before eluting the protein, unless the salts bind to the column too. This is unlikely for KCl, but it might happen with guanidine.

Have you considered using gel filtration chromatography to desalt your protein? This would allow you to remove most of the low molecular weight substances before performing the RPC. You could use this method with the protein that eluted from the RPC column after lyophilizing it and before reloading it. A simple and rapid desalting procedure can be performed on low-volume samples using PD-10 columns.

As I understand it you are suggesting to get rid of salts completely (mainly guanidine) by gel filtration chromatography?

It might be possible to desalt the sample before another round of RPC. However, our nucleoporin is a truncated version of FG domains. It is very hydrophobic and aggregates if no chaotropic agent is present. At low protein concentrations we can go as low as 2M Guanidine to keep them soluble (that's why we chose 2M GndHCL buffer for loading on the RPC column). Therefore, we need a certain amount of GndHCl to load a protein that is in a non-aggregated state.

I could buffer it into ACN buffer during gel filtration. This might work, but I never tested what exact ACN concentration is needed to keep the protein soluble at certain concentrations.

It might be as you said, that the guanidine somehow sticks to the column. As mentioned the protein loaded is two times higher than before, but the volume loaded is even 4 times higher than before (the previous sample was higher concentrated before loading on the PRC). Therefore, more GndHCl went through/on the column and might be bound on it.

It might be worth a try and repeat the RPC by taking the current batch up into the same loading buffer and performing an extensive wash after loading?

Thanks again for your feedback Adam B Shapiro !

An approach to returning the guanidine concentration to the desired 2 M value would be to dialyze the protein against a 2 M guanidine solution. However, this would use a lot of guanidine, since the dialysis buffer volume is much larger than the sample volume.

An alternative would be to equilibrate a gel filtration column (such as a PD-10 desalting column) with the 2M guanidine solution. Passing the sample through this column would result in the protein eluting in that solution, with excess guanidine eluting afterwards.

A third approach is to use diafiltration. The sample is concentrated with a centrifugal ultrafiltration device or ultrafiltration pressure cell. The low-molecular-weight salts pass through the membrane and the protein is retained.Then the protein is diluted with the desired solution and concentrated again. Make sure the ultrafiltration membrane chosen is compatible with concentrated guanidine.