The GxTX peptide used in surface labeling was synthesized at the Molecular Foundry of the Lawrence Berkeley National Laboratory under U.S. Department of Energy Contract DE-AC02-05CH11231. GxTX-633 was synthesized by conjugating GxTX to DyLight 633 Maleimide (ThermoFisher Scientific Cat# 46613) using methods for GxTX–maleimide conjugates described previously (Tilley et al., 2014 (link)). HEK293T cells were surface-labeled with 1 μM GxTX as described previously (Tilley et al., 2014 (link)) and imaged in TIRF as described below but in physiological saline solution (4.7 mM KCl, 146 mM NaCl, 2.5 mM CaCl2, 0.6 mM MgSO4, 1.6 mM NaHCO3, 0.15 mM NaH2PO4, 20 mM HEPES, pH 7.4) containing 8 mM glucose, 0.1 mM ascorbic acid, and 0.1% BSA.
Dylight 633 maleimide
Manufactured by Thermo Fisher Scientific
DyLight 633 Maleimide is a fluorescent label used for protein labeling. It is a maleimide reactive dye that can form a covalent bond with sulfhydryl groups on proteins. The DyLight 633 dye has an absorption maximum at 638 nm and an emission maximum at 658 nm, making it suitable for detection in the red/far-red region of the visible spectrum.
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Lab products found in correlation
Bilirubin oxidase, by Merck Group (1 mentions)
Unconjugated bilirubin, by Merck Group (1 mentions)
Pefablock, by Merck Group (1 mentions)
Seakem hgt p agarose, by Lonza (1 mentions)
Rabbit anti human vwf antibody, by Agilent Technologies (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Typhoon 9200 imager, by GE Healthcare (1 mentions)
Typhoon 9400 imager, by GE Healthcare (1 mentions)
4 protocols using dylight 633 maleimide
1
Surface Labeling of HEK293T Cells
2
Unconjugated Bilirubin Binding Assay
3
Fluorescent Labeling of RbpA Protein
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Purified RbpA protein was conjugated with the sulfhydryl-reactive dye, DyLight633 maleimide (Thermo Scientific), at the single cysteine site (C56) according to the manufacturer protocol. Briefly, 1 mg of RbpA protein was incubated with the 20 μl maleimide-activated dye in the conjugation buffer (0.1 M phosphate, 0.15 M NaCl, 2 mM EDTA, pH 7.4) at 24°C for 2 h. The excess dye reagent was then removed from the sample by dialysis. Protein was concentrated to 4 mg/ml by Ultracel-10 membrane filter unit (Millipore) and stored at −20°C in 50% glycerol. For the native gel analysis, the labeled RbpA (1.6 μM) was incubated with 2.4 μM of indicated σ subunit in 10 μl TB at 37°C for 15 min. The complexes were analyzed on 5–10% native PAGE in Tris–Glycine buffer. Gels were scanned by Typhoon 9200 Imager (GE Healthcare) and quantified using ImageQuant software (Molecular Dynamics).
4
Fluorescent Labeling of RbpA
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RbpA was conjugated with the sulfhydryl- reactive dye, DyLight 633 Maleimide (Thermo Scientific), as described (24 (link)). Labeled RbpA (1.6 μM) was incubated with different concentrations of the σB subunit (0.8, 1.6 and 3.2 μM) in 10 μl of TB at 37°C for 10 min. Samples were analyzed on 5–10% native PAGE in Tris–glycine buffer. Gels were scanned with a Typhoon 9400 Imager (GE Healthcare).
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