Hi,

I am reaching out to you because I have a problem that has remained unresolved for far too long.

I want to label my intrinsically disordered protein with a nitroxide probe. My WT protein does not contain any cysteine, so I created a mutant containing cysteine instead of alanine.

The production and purification of my protein are complicated because it is hydrophobic and sticks to all plastics, resulting in a fairly low yield at the end.

In addition, it is not very stable, so I have to add 1X protease inhibitor to prevent it from degrading too quickly. Only one of the compounds in the cocktail is a protein that contains cysteine. So I don't have to add any, and my protein can last for about a week. Its optimal buffer is NaPO4/NaCl at pH 6.

First, I tried to label the protein following the usual protocol, i.e.:

1- DTT reduction

2- removal of excess by PD10 column

3- incubation with excess 20 molar Proxyl at 4°C

4- removal of excess proxyl by PD10 column

Except that at the end, there was no more protein...

So I followed this protocol, replacing the PD10 columns with dialysis: 3 dialysis sessions of 2 hours + one O.N. dialysis.

It works! My protein is still there at the end of the labelling process.

I observed the effect of the labelling on my protein by NMR in solution, but to my surprise, there was not much effect.

I contacted EPR specialists who are familiar with this labelling protocol. They taught me how to quantify the proportion of labelled protein relative to unlabelled protein in my final fraction.

Surprisingly for an IDP, the labelling percentage did not exceed 50%.

We considered several modifications to the protocol.

A labelling pH of 7 would be more optimal, incubation temperature at RT, and excess proxyl only at 10 molar.

On the recommendation of a colleague, we added 2M urea to stabilise the protein and prevent it from sticking to the Amicon columns and membrane. This works really well, and we can use desalting columns and Amicon columns.

With all these changes, we are still unable to label the protein...

Have you encountered the same kind of problem? Do you have any ideas or suggestions?