Expansion of a CAG triplet repeat within the first exon of the HUNTINGTIN gene encoding for a polyglutamine tract is the cause of a progressive neurodegenerative disorder known as Huntington's disease. N-terminal fragments of mutant huntingtin have a strong propensity to form oligomers and aggregates that have been linked to the Huntington's disease pathology by different mechanisms, including gain of toxic functions. The biological and biophysical properties of the polyglutamine expansion within these huntingtin fragments are influenced by neighboring domains, in particular by the first 17 amino acids of huntingtin (N17), which precede the polyglutamine expansion. It has been suggested that N17 phosphorylation modulate mutant huntingtin aggregation and toxicity, but the study of its functional and pathological relevance requires the capacity to detect this modification in biological samples in a simple, robust way, that ideally provides information on the abundance of a phosphorylated species relative to the total pool of the protein of interest. Using a modified SDS-PAGE protocol (Phos-Tag) followed by Western blotting with specific anti-HUNTINGTIN antibodies, we efficiently resolved huntingtin fragments expressed in cellular contexts based on the presence of phosphorylated residues, we defined threonine 3 as the major site of huntingtin N17 phosphorylation and, finally, we identified IKK-beta as a kinase capable of phosphorylating threonine 3 in N-terminal hungtingtin fragments. (C) 2015 Elsevier Inc. All rights reserved.