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Cloning and Purification of PNKP FHA Domain: Enzyme Digestion and Western Blot Analysis

This study focuses on the cloning and purification of the PNKP FHA domain. We present a detailed diagram of the PNKP cDNA and demonstrate the efficient serial restriction enzyme digestion using BamHI and XbaI. The purification of the His-tagged FHA domain was successfully achieved using Ni-NTA chromatography. Furthermore, we conducted a thorough western blot analysis using both monoclonal and polyclonal antibodies, confirming the detection of the FHA domain. This work provides valuable insights into PNKP domain characterization for further studies.

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Cloning and Purification of PNKP FHA Domain: Enzyme Digestion and Western Blot Analysis

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  1. A XbaI 10xHis FHA domain BamHI Phosphate domain Kinase domain Bam HI 5’- -3’ C F10 B Lysate FL W1 W2 F1 F2 F3 F4 F5 F6 F7 F8 F9 62 Bam HI/XbaI Bam HI Marker 47.5 32 10..0K 25 8.0K 6.0K 5.0K 4.5K 16.5 4.0K 3.5K 3.0K 2.5K 2.0K D antiPNK 1.5K 1.0K monoAb PolyAb 0.5K 0.25K 16.5K Supplemental figure 1. Cloning and purification of PNKP FHA domain. (A) Diagram of the PNKP cDNA; (B) serial restriction enzyme digestion of PNKP cDNA with BamHI and XbaI; (C) chromatography of His-tagged FHA domain on Ni-NTA column; (D) western blot analysis showing both monoclonal and polyclonal antibodies of PNKP can detect FHA domain.

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