Journal: Methods in enzymology

Article Title: A quick and colorful method to measure low-level contaminations of paramagnetic Ni 2+ in protein samples purified by immobilized metal ion affinity chromatography

doi: 10.1016/bs.mie.2018.08.037

Figure Lengend Snippet: Ni2+ leakage from three commonly used IMAC resins. (A) A column containing 10 mL of pristine His•Bind resin was rinsed with column volumes 3 (CV) of milliQ water, charged with 5 CV of 50 mM NiSO4, and rinsed with 5 CV of binding buffer (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.9). The column was then (a) equilibrated with an additional 10 CV of binding buffer, (b) washed with 6 CV of wash buffer (20 mM Tris-HCl, 500 mM NaCl, 60 mM imidazole, pH 7.9), and (c) eluted using a 60 – 1000 mM imidazole in 20 mM Tris-HCl, 500 mM NaCl, pH 7.9, gradient. Panel A displays the elution profile of steps a to c. (B) A column containing 10 mL of pristine Ni2+ pre-charged His60 Ni Superflow resin was rinsed with 10 CV of equilibration buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 7.4). The column was then (a) washed with an additional 10 CV of equilibration buffer, (b) washed with 10 CV of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 40 mM imidazole, pH 7.4), and (c) eluted using a 40 – 300 mM imidazole in 50 mM NaH2PO4, 300 mM NaCl, pH 7.4, gradient. Panel B displays the elution profile of steps a to c. (C) A column containing 10 mL of pristine Ni2+ pre-charged cOmplete His-Tag Purification resin was rinsed with 10 CV of equilibration buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The column was then (a) washed with an additional 10 CV of equilibration buffer and (b) eluted using a 0 – 250 mM imidazole in 50 mM NaH2PO4, 300 mM NaCl, pH 8.0, gradient. Panel C displays the elution profile of steps a and b.

Article Snippet: To refine the α 3 X purification protocol and minimize Ni 2+ co-purification, we investigated Ni 2+ leakage from three IMAC products: His•Bind resin (Novagen; chelator chemistry IDA, stationary matrix agarose, binding capacity 8 mg protein/mL resin), Ni 2+ preloaded His60 Ni Superflow resin (Clontech; chelator chemistry IDA, stationary matrix Superflow 6 agarose beads, binding capacity ≤ 60 mg protein/mL) and Ni 2+ preloaded cOmplete His-Tag Purification resin (Roche; chelator chemistry proprietary; stationary matrix Sepharose-CL 6B, binding capacity ≥ 40 mg protein/mL resin).

Techniques: Binding Assay, Purification

Journal: American Journal of Cancer Research

Article Title: Proscillaridin A inhibits lung cancer cell growth and motility through downregulation of the EGFR-Src-associated pathway

doi:

Figure Lengend Snippet: Effect of proscillaridin A on cell viability and clonogenicity in NSCLC cell lines with different EGFR statuses. A. Proscillaridin A cytotoxicity determined by cell viability assay of NSCLC cell lines with different EGFR statuses. These results are presented as percentages compared with the vehicle control (0 µM, 0.1% DMSO). The I.C. 50 at each time point is shown at the bottom of each bar chart. Each experiment was independent and repeated three times. B and C. Clonogenicity was determined by colony formation assay without or with LMP agarose. B. Anchorage-dependent colony formation (without agarose). Colonies with diameters ≥ 0.5 mm were counted. C. Anchorage-independent colony formation (with agarose). Colonies with diameters ≥ 0.5 mm were counted. Each experiment was independently performed in triplicate; 0 nM: 0.1% DMSO. *P < 0.05 compared with the vehicle control.

Article Snippet: Briefly, cells were treated with proscillaridin A for 48 hrs, and equal volumes of cell lysates were incubated with 2 μg of homemade GST-tagged p21-binding domain (GST-PBD) fusion protein preloaded on glutathione sepharose 4B resin (GE healthcare, Pittsburgh, PA, USA) to pull down active Rac1 and Cdc42 protein at 4°C for 1 hr.

Techniques: Viability Assay, Colony Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Genome-wide YFP Fluorescence Complementation Screen Identifies New Regulators for Telomere Signaling in Human Cells *

doi: 10.1074/mcp.M110.001628

Figure Lengend Snippet: Confirmation of protein-protein interactions identified by the screens. A, Fluorescent microscopy images of bait-prey protein pairs tagged with YFP fragments. HTC75 cells stably coexpressing YFPn-tagged TRF1 and YFPc alone (negative control), YFPc-tagged TIN2 (positive control), YFPc-MDH1, or YFPc-SET were visualized live under a fluorescence microscope. Hoechst33342 was added to visualize the nuclei. B, A flow chart of secondary coprecipitation screens that were used to confirm the identified protein-protein interactions. C, Examples of coprecipitation screens. Cell extracts from 293T cells coexpressing FLAG-tagged candidate proteins with GST alone or GST-tagged telomeric bait proteins were incubated with GSH-agarose beads. The proteins bound to GSH beads were then dot-blotted and probed with anti-FLAG antibodies.

Article Snippet: The cleared lysate was then transferred into a 96-well binding plate (Purelink Clarificaiton plate, Invitrogen) preloaded with 100 μl/well of glutathione Sepharose 4B beads (10% slurry) (GE Healthcare Bio-Sciences AB), and incubated for 2 h at 4 °C with gentle agitation.

Techniques: Microscopy, Stable Transfection, Negative Control, Positive Control, Fluorescence, Incubation