Fig 1.
Expression and purification of ZF arrays.
A. E. coli transformed with plasmids encoding His-, GST-, and His-SUMO-tagged CXCR4L (gel lanes labeled “His,” “GST,” and “SUMO,” respectively) before (“–”) and after (“+”) IPTG induction were loaded on a denaturing polyacrylamide gel and stained with Coomassie Blue. Black asterisk and black arrows indicate the position of the tagged ZF arrays. B. Supernatant (S) and pellet (P) fractions of a whole cell lysate containing GST-CXCR4L were separated on a denaturing polyacrylamide gel and stained with Coomassie Blue (left panel) or transferred onto a PVDF membrane and detected with an anti-GST antibody (right panel). C. Comparison by SDS-PAGE and western blot (anti-His) of resuspended Ni-NTA resins bound to His-CXCR4L (His, black arrows on the left) or His-SUMO-CXCR4L (“SUMO”, black arrows on the right). For each tagged form, the resin volume was kept consistent throughout the procedure. D. Schematic representation of the purification protocol. E. Samples from each step of the purification protocol were separated on a denaturing polyacrylamide gel and stained with Coomassie Blue. S: Supernatant of cell lysate; FT: Ni-NTA flow-through; W: Ni-NTA washes; R: Ni-NTA resin resuspended in cleavage buffer; E: recovered fraction after overnight cleavage. F. Samples spanning the main peak of the cation-exchange chromatographic elution were separated on a denaturing polyacrylamide gel and stained with Coomassie Blue.
Table 1.
Zinc finger arrays used in this study.
Fig 2.
Effect of L-arginine on Ni-NTA elution.
Analysis by SDS-PAGE of the same steps in presence or not of L-arginine. BC: resin before addition of Ulp1; AC: resin after overnight cleavage; E: recovered fraction after overnight cleavage; R: resin after elution.
Fig 3.
A general method for the purification and labeling of ZF arrays.
Eight ZF arrays purified by the two-step method outlined in Fig 1 and labeled with maleimide-activated sulfo-Cy5 were separated, alongside molecular weight markers (M), on a denaturing polyacrylamide gel. A. Top: the gel after staining with Coomassie Blue. Bottom: the same gel upon illumination with 650-nm light. B. Cy5- and Cy3-labeled TZAP9–11 were electrophoresed through a denaturing polyacrylamide gel and imaged under 650-nm and 532-nm illumination.
Fig 4.
TZAP9–11 binds telomeres in situ.
Fixed U2OS cells stained with DAPI (left, blue in the merge image) and incubated with Alexa-Fluor-647-labeled anti-TRF2 (red in the merge image) and Cy3-TZAP9–11 (green in the merge image) were imaged on a widefield microscope. Colocalization (yellow) of Cy3-TZAP9–11 and anti-TRF2 confirms telomeric localization of TZAP9–11. Scale bar: 10 μm.
Fig 5.
Affinity and specificity of ZF arrays for DNA, measured by MST.
Each panel shows a binding curve involving the ZF array indicated in the bottom right corner and wild-type (green) or point-mutant (red) DNA sequences. The ordinate represents fractional saturation and the abscissa represents the concentration of the oligonucleotide. Solid lines represent fits to a non-cooperative 1:1 binding model.