Hello all,

I'm trying to determine the ubiquitin linkage site of a polyubiquitinated protein using HA-tagged ubiquitin mutants (K0, K48, K63, K48R and K63R). For this I'm immunoprecipitating my protein of interest, and blotting for HA.

However, my input blot is showing different levels of polyubiquitination from each of my mutant constructs, suggesting differences in construct stability. This is likely to make interpreting the pulldown data difficult.

Previous threads have suggested comparing the levels of mono-ubiquitin in the input, and adjusting the transfection ratios until these are equal. However, despite re-running my samples on a gradient gel, I'm unable to detect any non-conjugated monomeric HA-Ub species around the 10 kDa marker.

Does anyone have an idea why this could be? Any tips and trips would be appreciated.

Thanks