Journal: Nature methods

Article Title: SNAC-tag for Sequence-specific Chemical Protein Cleavage

doi: 10.1038/s41592-019-0357-3

Figure Lengend Snippet: a. TEV protease cleavage of HisTag-ENLYFQS-HB2225 construct, cleavage conditions: protein 1 mg/mL, 50 mM Tris, pH 8.0, 0.5 mM EDTA, 1 mM DTT, 0.25 M NaCl, 22 °C, 16 hours, TEV protease 0.04 mg/mL. b. Ni 2+ cleavage of HisTag-GSHHW-HB2225 construct, cleavage conditions: protein 1 mg/mL, 1.0 mM NiCl 2 , 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, pH 8.2, 22 °C, 16 hours. c. Cleavage compatibility test for Ni 2+ cleavage of HisTag-GSHHW-HB2225. d. TEV protease cleavage of HisTag-T4L-ENLYFQS-3hbtmV2 construct, cleavage conditions: protein 1 mg/mL, 50 mM Tris, pH 8.0, 0.5 mM EDTA, 1mM DTT, 0.25 M NaCl, 22 °C, 5 mM DPC, 16 hours, TEV 0.04 mg/mL. e. Ni 2+ cleavage of HisTag-T4L-GSHHW-3hbtmV2 construct, cleavage conditions: protein 1 mg/mL, 1.0 mM NiCl 2 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, pH 8.2, 22 °C, 5 mM DPC, 16 hours. f. Cleavage compatibility test for Ni 2+ cleavage of HisTag-T4L-GSHHW-3hbtmV2. g. Ni 2+ cleavage of HisTag-T4L-GSHHW-3hbtmV2 in different detergents. h. Cleavage of the membrane protein HisTag-T4L-XXXXXXXX-3hbtmV2. Lane 1 shows protein before cleavage, lanes 2 to 14 shows cleavage using difference sequences from phage selections, the sequence inserted for each lane is shown on top of the lane. i. Cleavage of the globular protein construct HisTag-XXXXXXXX-HB2225, lane 1 shows the position of the protein before cleavage, lane 2 to lane 13 shows cleavage using difference sequences from phage selection, the sequence inserted for each lane is shown on top of the lane. Cleavage percentages were quantified based on cleaved and uncleaved protein bands in each individual lane in Imagelab 5.2.1 and is summarized in . Full gels are shown in .

Article Snippet: Cleavage efficiency were quantified using Imagelab 5.2.1 on Bio-Rad ChemiDoc MP imaging System.

Techniques: Construct, Membrane, Sequencing, Selection

Journal: Molecular and Cellular Biology

Article Title: LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia

doi: 10.1128/MCB.00652-19

Figure Lengend Snippet: Reagents and resources

Article Snippet: Catalog no. S7579 pBluescript SK Stratagene Iscove's modified Dulbecco's medium (IMDM) Gibco Catalog no. 12200-036 RPMI 1640 Gibco Catalog no. 31800-022 Penicillin-streptomycin solution 10× Corning Catalog no. 30-022-CI Geneticin Gibco Catalog no. 10131-027 0.05% trypsin, 0.53 mM EDTA 1× [−]sodium bicarbonate Corning Catalog no. 20116004 Puromycin dihydrochloride Fisher Bioreagents Catalog no. BP2956-100 Pierce protease inhibitor tablets Thermo Scientific Catalog no. A32965 Hygromycin B-PBS (50 mg/ml) Invitrogen Catalog no. 10687010 Anti-FLAG M2 resin Sigma Catalog no. A2220 Protein A/G resin Santa Cruz Polyvinylidene difluoride (PVDF) membrane GE Catalog no. 10600022 SuperSignal PicoWest Plus Thermo/Pierce Catalog no. 1863099 Experimental models: cell lines Human: HEK 293 ATCC Human: Jurkat ATCC Human: K562 ATCC Human: KOPTK1 ATCC Human: LOUCY ATCC Human: U937 ATCC Human: U2OS C32 Halo-CTCF Hansen et al. ( 27 ) U2OS endogenous knock-in cell line where all endogenous copies of CTCF have been N-terminally tagged with FLAG-HaloTag; clone 32 Software and algorithms Flowjo 10.3 analysis software FlowJo, LLC Contact corresponding author for URL Ideas software Amnis Corporation Contact corresponding author for URL ImageLab 5.2.1 BioRad Contact corresponding author for URL Imaris Bitplane Inc. Other CytoFLEX benchtop cytometer Beckman Contact corresponding author for URL Leica TCS SP8 confocal imaging system Leica Contact corresponding author for URL ImageStream Mk II Amnis Contact corresponding author for URL Open in a separate window Reagents and resources

Techniques: Recombinant, Staining, Modification, Protease Inhibitor, Membrane, Knock-In, Software, Cytometry, Imaging