lambda protein phosphatase λpp based dephosphorylation assay  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Autophosphorylation of the Tousled-like kinases TLK1 and TLK2 regulates recruitment to damaged chromatin via PCNA interaction

doi: 10.1093/nar/gkae1279

Figure Lengend Snippet: Tousled-like kinases are autophosphorylated at their N-terminus. ( A ) Co-transfection of TLK1 wildtype and catalytic inactive mutant D607A with the indicated amount of plasmids in HEK293T cells followed by Western blotting analysis. ( B and C ) Kinase assays using ATP-γ-S followed by alkylation by p-Nitrobenzyl mesylate to convert phosphorylated residues into thiophosphate esters. Antibody against thiophosphate ester was used to identify phosphorylation events. ( D and E ) Lambda protein phosphatase (λpp) assay was performed using SFB-tagged TLK1 wildtype and D607A mutant or SFB-tagged TLK2 wildtype and D613A mutant purified from HEK293T cell lysate. Differential molecular weights were analyzed by Western blotting analysis. ( F ) TLK1 is autophosphorylated at nine residues within its N-terminus. Phospho-Tandem-Mass-Tag mass spectrometry was performed on phosphopeptides enriched from HEK293T cells with TLK1 overexpression and knockdown using siRNA. ( G ) TLK1 N-terminus is highly phosphorylated. SFB-tagged TLK1 wildtype, D607A or Δ1–240 (ΔN-terminus) were purified from HEK293T using streptavidin beads followed by Western blotting analysis using phospho-Serine and phospho-threonine antibodies. ( H ) TLK1 wildtype, N-terminal 9S/T to A and catalytic inactive mutants were treated with λ-phosphatase. Differential molecular weights were analyzed by Western blot.

Article Snippet: SFB-tagged proteins were aliquoted into a fresh 1.5 ml tube containing 1× NEBuffer for Protein MetalloPhosphatases supplemented with 1 mM MnCl 2 , and 400 units of Lambda protein phosphatase (λpp) (New England Biolabs, P0753S).

Techniques: Cotransfection, Mutagenesis, Western Blot, Purification, Mass Spectrometry, Over Expression, Knockdown