How much TCEP are you using? I know it is promoted as being maleimide- friendly, but it certainly does interfere at fairly modest concentrations. If you have no issues with TCEP, the molar ratio of peptide to linker is going to be an important parameter. What ratio did you try? Finally, your product is relatively small, are you sure your SDS-gel is capable of differentiating the monomer and dimer?

Nick Gee

I'm using 5mM TCEP. The linker is used in a 2-fold molar excess to the peptide/protein, final concentrations are 0,2mM linker and 0,1mM peptide/protein. We use Bis-Tris 4-12% gels so the bands should be dicernable. At least with lysozyme it should be clearly visible, but that didn't work either.

But I plan to try different ratios and work with bigger proteins, probably the lysozyme, until I find a good protocol.

Hi Claire,

Means you have 4x excess of linker vs. dipeptide.

I think your problem can be resolved by optimization:

Try a slight excess of monomers, add the linker in aliquots and extend the reaction time. Also try if less TCEP. (0.5 to 1mM) works. Follow by SDS-PAGE or better HPLC (needs less material).

Instead of DTT to quench, you could use Cys. To be on the safe side, check your reaction on a gel also before adding the quencher, as the maleimide-thiol reaction is somewhat reversible (for more on this, take a look here: https://www.iris-biotech.de/blog/maleimide-crosslinkers/)

What is the pH of your reaction?

Wolfgang Schechinger

Thanks, I'll try your suggestions!

I currently work with a pH of 7.4 but I was thinking of trying acidic pH, because I read it helps avoid thiazine formation.

Yes, your ratio is suboptimal on theoretical grounds. I don’t think there is much wrong with the pH at 7.4, but the reaction will still work at lower pH values. Whatever pH is used, you do need the peptide in molar excess over the maleimide functions to get the desired product in high yield. A better approach than manipulating pH to stop the N-terminal amine from triggering the side reaction is simply to block the amine.

I'll try the different approaches, thanks!