I have been struggling with CuACC in HeLa lysates for months. I am using an activity-based alkyne probe which becomes attached to proteins. I click the tag to biotin-azide for visualization on a gel. With recombinant protein, the reaction works as expected, producing copper-dependent signal, and all my negative controls look good.

In lysates, however, I observe a strange paradox. On my gels, I only ever see signal in the absence of copper. That's right, with HeLa lysate, probe, biotin-azide, TCEP, and TBTA, I see a low level of signal. But once I add copper, the signal goes away completely. I've remade every reagent, tried cysteine-capping the lysates with iodoacetamide, making up new lysates, varying the Cu/TBTA ratio, etc.

The only thing I've learned for sure is that this signal loss is copper-dependent. The more copper I add, the less signal I see. I've tested 0.001 - 1 mM CuSO4, and 0.001 gives similar signal to no copper at all, and the signal decreases linearly until about 0.5 mM, when it disappears completely.

At this point, I'm of the opinion that some side reaction is occurring rapidly in a copper-dependent fashion, consuming all the label on my protein before it can react with my biotin azide. However, I cannot figure out what this side reaction could be or how to avoid it. If anyone has experience here I'd greatly appreciate it, this has delayed the completion of this project by three months already.

Some specs:

My reaction buffer (1x) is 50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1 mM KCl.

The lysis buffer my cells are in is 50 mM Tris HCl pH 7.4, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100. (Lysate is diluted in the reaction 3-4 fold). I have also tried running my lysate through a 3 kDa filter in PBS to remove small molecule thiols and other potential small molecule interferants to no avail.

The concentrations of my click rxn (final) are 1.3 mM CuSO4, 0.13 mM TBTA, 2 mM TCEP, 2mM biotin-azide, following a reaction with my probe (1 mM alkyne). I have varied these parameters considerably - I have tried Cu/TBTA between 10:1 and 1:5, I have tried TCEP concentration 1 - 5 mM.

The attached image is a result of a typical experiment. Lane 1 is ladder, Lane 2 is background (lysate, click reagents, buffer without alkyne probe), Lane 3 is experimental (lysate, click reagents, buffer, probe), Lane 4 is no copper control (lysate, click reagents sans copper, buffer, probe), Lane 5 is an inhibitor control lane (lysate, click reagents, buffer, probe, probe inhibitor).