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. Author manuscript; available in PMC: 2017 Nov 20.
Published in final edited form as: Mol Plant. 2017 Apr 28;10(6):846–865. doi: 10.1016/j.molp.2017.04.008

Figure 1. Two-Step Affinity Purification of Ubiquitylated Proteins from Etiolated Arabidopsis Seedlings Before and After Red-Light Irradiation.

Figure 1

Ub conjugates were affinity purified by sequential TUBEs (1) and Ni-NTA (Ni) chromatography (2) steps from 7-day-old 6His-UBQ seedlings either kept in the dark (D) or exposed to 1 h of red light (D-R). Samples from dark-grown wild-type (WT) seedlings, which were subjected to the same purification protocol using GST and Ni-NTA beads, were included for comparison.

(A) SDS–PAGE analysis of the various fractions during the purification. The gels were either subjected to immunoblotting with anti-Ub antibodies (@Ub) or stained for protein with silver (Protein). FT and EL represent flow through and elution fractions, respectively. Extract represents the initial clarified seedling extract. Ub conjugates and the Ub and 6His-Ub monomers and dimers are indicated.

(B) The two-step affinity purification also enriches for PhyA-Ub conjugates that accumulate in etiolated seedlings after R. The samples analyzed in (A) were immunoblotted with the monoclonal antibody 073D against PhyA.