Particles were incubated in human plasma (1.83E7/mL) or buffer for 1 hour at 37°C, as done previously.[34 (link)] Then, particles were washed 3x in PBS −/− and resuspended in 50uL SDS stain (Pierce SDS-PAGE Sample Prep Kit). Particles were placed in a Bio-Rad T100 Thermal Cycler and boiled to strip proteins from the particle surface. Particles were then centrifuged and the supernatant solution was run on a Novex WedgeWell 4-20% Tris-Glycine Gel alongside a protein ladder. Gels were then stained using EasyBlue Safe Stain, and destained by shaking overnight in deionized water. Images were taken using a Bio-Rad EZ Gel Imager. Specific bands were cut out, digested, and analyzed via LCMS at the University of Michigan proteomics core.
Wedgewell 4 20 tris glycine gel
Manufactured by Thermo Fisher Scientific
The WedgeWell 4–20% Tris-Glycine gel is a pre-cast polyacrylamide gel used for protein separation and analysis. It features a continuous 4–20% gradient of acrylamide concentration, which allows for the separation of a wide range of protein molecular weights.
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4 protocols using wedgewell 4 20 tris glycine gel
1
Protein Profiling of Nanoparticles in Plasma
2
Quantitative Profiling of Proteome Modifications
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Live T-47D cells were plated on 6 cm petri dishes supplemented with RPMI media and incubated for 24 h. Cells were then treated with probe 1i (100 µM to 2 mM), CuBr (100 µM to 2 mM), and 1% acetonitrile for 2 h. After 2 h, cells were washed 3 times with cold PBS and lysed using RIPA buffer (50 mM Tris HCl [pH 8], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors. Lysates were centrifuged 6500x g, 10 min at 4 °C, and soluble lysate was collected. To 100 µg of lysate in 100 µL of PBS buffer were treated with 50 µL of 100 mM TBTA in water, 50 µL of freshly prepared 100 mM ascorbic acid in water, 50 µL of 50 mM of CuSO4 in water, and 2 µL of 10 mM Cy5 azide in DMSO. The reaction was stirred for 1 h and acetone precipitated, followed by analysis of proteins through in-gel fluorescence imaging and Coomassie blue staining. Samples were loaded on a Novex WedgeWell 4–20% Tris-Glycine gel. Gel was run in Tris-glycine running buffer at 180 V. The gel was then stained with Coomassie brilliant blue for 1 h and destained overnight. For proteomics analysis, 100 µg of live-cell derived lysates were digested using SMART Digest™ Trypsin Kit by Thermo Scientific. Proteomics analysis was done according to the general protocol described above.
4
Copper-Mediated Protein Labeling and Profiling
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To 4 tubes (individual reactions) of 100 µg of lysate in degassed MeCN:H2O (1:4, 400 µL) were treated with freshly prepared 50 µM, 100 µM, 150 µM, and 200 µM CuBr that had been re-suspended in 50 µL of acetonitrile. To the 4 samples were added freshly prepared 50 µM, 100 µM, 150 µM, and 200 µM probe 1i that had been re-suspended in 100 µL of NaP buffer pH 7. The reaction was stirred at room temperature for 3 h. Upon completion of reaction, samples were acetone precipitated, followed by Cy5 azide fluorophore labeling. Proteins were dissolved in 100 µL of water, followed by the addition of 50 µL of 100 mM TBTA in water, 50 µL of freshly prepared 100 mM ascorbic acid in water, 50 µL of 50 mM of CuSO4 in water, and 2 µL of 10 mM Cy5 azide in DMSO. The reaction was stirred for 1 h and acetone precipitated, followed by analysis of proteins through in-gel fluorescence imaging and Coomassie blue staining. Samples were loaded on a Novex WedgeWell 4–20% Tris-Glycine gel. Gel was run in Tris-glycine running buffer at 180 V. The gel was then stained with Coomassie brilliant blue for 1 h and destained overnight.
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