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. 2012 Aug 17;7(8):e43477. doi: 10.1371/journal.pone.0043477

Figure 5. In vitro ubiquitylation assay of PfUBA1 and PfUBC.

Figure 5

(A) 6xHIS-tagged recombinant PfUBA1 (E1) and PfUBC (E2) are depicted and anti-HIS blots reveal purification. (B) In vitro ubiquitylation assays were performed with recombinant PfUBC (E2), which revealed an attachment of a single ubiquitin when incubated with human UBA1 (E1) (second lane from the right) and an increased number of ubiquitylation products when both UBA1 and recombinant PfHRD1(E3) was added (far right lane). (C) When incubated with human UBA1 (E1), recombinant PfUBC (E2) (along with the other necessary reagents) attaches polymers of ubiquitin to recombinant PfHRD1 (E3) (far right lane). (D) Recombinant PfUBA1 (E1) is capable of attaching a single ubiquitin to recombinant PfUBC (E2), which is depicted by the appearance of a 27 kDa band (second lane from the right). Extra banding was not detected with the addition of recombinant PfHRD1 (E3) (far right lane).