K6-linked Ubiquitylation Marks Formaldehyde-Induced RNA-Protein Crosslinks for Resolution. Suryo Rahmanto, Blum et al.
The files provided in this dataset contain uncropped original western blots published in the "K6-linked Ubiquitylation Marks Formaldehyde-Induced RNA-Protein Crosslinks for Resolution" by Suryo Rahmanto and Blum et al.
Steps to reproduce
For general Western blotting, whole cell lysates (WCL) were collected in modified RIPA buffer, which had been supplemented with complete protease inhibitor. WCL were clarified by sonication and centrifugation to remove insoluble lysate materials. Soluble proteins were subsequently quantified by Bio-Rad quick protein assay reagent and equal protein amounts were loaded onto 4-12% Bis-Tris SDS-PAGE for gel electrophoresis. Separated protein lysates were next blotted onto PVDF membrane and probed with designated primary antibody at 4 oC overnight. Primary antibodies are prepared in PBS buffer containing 3% (w/v) BSA and 0.1% (v/v) Tween-20. Primary antibodies are visualized by chemiluminescence using the appropriate HRP-conjugated secondary antibodies, whereby the digitized data is acquired using Bio-Rad Chemidoc Imager system. For a diK6-ubiquitin pull-down experiment, K6-specific probes (i.e. Biotin-conjugated diK6 affimer or LotA-N) were added to clarified lysates. Samples were left rotated at 4 oC for a minimum of 2 hours prior to subsequent pulldown using High-capacity NeutrAvidin agarose beads. Following the agaraose pulldown, unbound proteins were washed away by multiple washing with modified RIPA buffer. Proteins bound to the beads were finally eluted in 2x LDS buffer and reduced with DTT prior to gel electrophoresis and Western blotting as described above.