Micro bio spin chromatography column

Firstly, 0, 0.25, or 0.5 μM MBP-TEV-DDX3X or MBP-TEV-DDX3Y in buffer (200 mM NaCl, 25 mM Tris-HCl pH 8.0) was incubated in TDP-43 assay buffer (150 mM NaCl, 20 mM HEPES-NaOH pH 7.0, 1mM DTT) with 0.5 μg TEV protease for 30 minutes at room temperature. Turbidity was assessed by measuring absorbance at 395 nm in a Tecan plate reader. TDP-43 was buffer exchanged into TDP-43 assay buffer (Bio-Rad Micro Bio-Spin Chromatography Columns, following manufacturer’s instructions) and concentration was determined via NanoDrop. Turbidity measurements were paused after 30 minutes in order to add 4.0 μM TDP-43 (or an equal volume of TDP-43 assay buffer) to the reaction. Then, turbidity measurements were resumed for an additional 16 hours. The data was standardized by subtracting out the initial reading at t = 1 min. from each respective condition. Values from conditions with DDX3X or DDX3Y alone were then subtracted from the appropriate conditions with TDP-43. Area under the curve analysis was used to compare the extent of aggregation for each condition. The t_1/2 of aggregation was determined by performing a nonlinear regression (asymmetric sigmoidal) on the data starting after TDP-43 addition (t = 31 min.) (GraphPad Prism).

The assay was conducted according to our published protocol (Longerich et al., 2009 (link)). Nuclei acid substrates used in this study are reported in Table S1. Duplex DNA, R-loop and D-loop substrates were prepared by annealing of DNA and/or RNA oligos (IDT). Following electrophoresis in a native 12% acrylamid (29:1) TBE gel at room temperature in 1X TBE buffer, substrates were eluted from gel slices by dialysis and concentrated by centrifugation using Micro Bio-Spin Chromatography Columns (Bio-Rad). All the reactions contain 10 nM ³²P-labeled substrates. The RNase inhibitor RNaseOUT (Thermo Fisher) was added to reactions (0.16 units/ul, 1:250) containing RNA substrates. Reactions were run on native 5% acrylamid (29:1) TBE gels at 4°C. The gels were dried onto Whatman DE81 paper and analyzed in a Personal Molecular Imager FX PhosphorImager (Bio-Rad). GelQuant.NET (http://biochemlabsolutions.com/GelQuantNET.html) software was used to quantify non-shifted substrate in each lane for calculation of the percentage of substrate bound.

Materials used included iodoacetamide, dithiothreitol, 1 M triethylammonium bicarbonate (TEAB), protease inhibitor cocktail, HA antibody conjugated agarose, Polybrene (Sigma-Aldrich, St. Louis, MO); trypsin (Promega Corp., Madison, WI); formic acid (HCOOH) (MERCK, Darmstadt, Germany); Strep-Tactin sepharose (IBA TAGnologies, Gottingen, Germany); D-biotin (Alfa Aesar, Karlsruhe, Germany); micro Bio-Spin chromatography columns (Bio-Rad, Hercules, CA); Gateway LR Clonase II Enzyme Mix Kit, fetal bovine serum, and Lipofectamine 2000 (Invitrogen, Carlsbad, CA).