The NIRF probe targeting CD68+ macrophages was generated by coupling an amine-reactive NIR fluorochrome (NHS ester), VivoTag 680 (excitation peak 665 ± 5 nm, emission peak 688 ± 5 nm) (Perkin-Elmer), to a rat anti-mouse CD68 antibody (AbDSerotec) according to manufacturer’s instructions. In brief, VivoTag 680 was dissolved in dry dimethyl sulfoxide (Sigma-Aldrich) at a concentration of 10 mg/ml. Prior to the labeling, the buffer of the antibody was exchanged by dialysis into conjugation buffer (50 mM carbonate/bicarbonate buffer, pH 8.5) using Slide-A-Lyzer dialysis cassettes (AbD Serotec) according to the protocol provided by the manufacturer. After buffer exchange, 30 μl of VivoTag 680 was added to the rat anti-mouse CD68 antibody. Following 1 h of incubation at room temperature protected from the light, the NIRF CD68 probe (approximately 151 kDa) was separated from free fluorescent dye (approximately 1 kDa) and antibody oligomers (larger than 300 kDa) by fast protein liquid chromatography using a Superdex 200 resin in a pre-packed 10/300 GL column (GE Healthcare). Probe concentration was determined using a BCA Protein Assay Kit (Uptima) according to the protocol provided by the manufacturer.
Superdex 200 resin
Manufactured by GE Healthcare
Sourced in United States
Superdex 200 resin is a size-exclusion chromatography medium designed for the separation and purification of proteins, peptides, and other biomolecules. It is a cross-linked agarose-dextran composite material that provides a wide range of separation capabilities for a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Bradford dye reagent, by Bio-Rad (2 mentions)
3h formic acid, by Moravek Biochemicals (2 mentions)
Ad 14c nad, by PerkinElmer (2 mentions)
Blue sepharose 6 fast flow, by GE Healthcare (2 mentions)
Dry dimethyl sulfoxide, by Merck Group (1 mentions)
Vivotag 680, by PerkinElmer (1 mentions)
Pet16b plasmid, by Merck Group (1 mentions)
Prism version 6 for windows, by GraphPad (1 mentions)
Bacto tryptone, by Thermo Fisher Scientific (1 mentions)
Sds page sample loading buffer, by Beyotime (1 mentions)
Amicon ultra 30k, by Merck Group (1 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
8 protocols using superdex 200 resin
1
NIRF Macrophage Targeting Probe Synthesis
2
Characterizing Core-Shell Silica Nanoparticles
3
Purification and Characterization of RelA
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DTT, BSA and antibiotics were purchased from Melford Laboratories; polyacrylamide-bis polyacrylamide [30% (w/v), 37:5:1], Bacto tryptone and yeast extract for culture media were purchased from Oxoid. Chelating fast flow resin and Superdex 200 resin were purchased from GE Healthcare; primers were purchased from Eurofins; restriction enzymes and E. coli strain K12 JM109 were purchased from New England Biolabs; pET16b plasmid was purchased from Merck Chemicals; E. coli RelA was expressed using a strain from the ASKA Clone library purchased from Shigen; E. coli MRE600 (C6) strain was purchased from NCTC. Francisella philomiragia was obtained from A.T.C.C.; mRNA was purchased from ATDBio. Unless stated otherwise all other reagents were purchased from Sigma–Aldrich or Fisher Scientific. Graphpad Prism version 6 for Windows was obtained from Graphpad Software.
4
Protein Purification by GPC
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Soluble protein was filtered through a 0.22 μm syringe filter to remove large particles and injected into a 500 μL sample loop, prior to fractionation by gel permeation chromatography (GPC) using Superdex 200 resin packed in a 10/300 column connected to an ÄKTATM prime plus chromatography system (GE Healthcare, Chicago, IL, USA). The column was routinely calibrated using commercial gel filtration calibration kits from 75 to 669 kDa (GE Healthcare, USA) and equilibrated with 10 mM HEPES-KOH, pH 7.5, 100 mM NaCl, at a flow rate of 0.4 mL min−1. Fractions of 0.8 mL were collected when elution volume was 6.6 mL and concentrated 10-fold using an Amicon Ultra 30K centrifugal filter unit (Merck Millipore, Darmstadt, Germany). Concentrated samples were further supplemented with SDS–PAGE sample loading buffer (Beyotime Biotechnology, Haimen, China) following the manufacturer’s instructions before SDS-PAGE and Western blotting.
5
Purification and Characterization of β-Glucosidase
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Lyophilized β-glucosidase from sweet almonds, buffers, and p-nitrophenyl-β-d -glucopyranoside (PNPGluc) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pre-packed HiTrap-Q anion exchange column, and Superdex 200 resin were from GE Healthcare Life Sciences (Piscataway, NJ, USA). Electrophoresis reagents of high purity were purchased from Bio-Rad (Hercules, CA, USA).
6
Purification and Characterization of FDH Variants
7
Protein Extraction and Fractionation Protocol
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Samples used for native-PAGE activity staining were extracted from three developing endosperms at 10 to 15 days after flowering following the methods of Miura et al.16) (link)
Soluble protein (SP), loosely-bound protein (LBP) and tightly-bound protein (TBP) fractions were extracted from three developing endosperms as described by Fujita et al.20) (link) Western blotting was performed as described previously.21) (link)
A 700 mg aliquot of developing endosperms was extracted and fractionated by gel filtration chromatography using Superdex 200 resin packed in a XK16 column (16/400, GE Healthcare, Chicago, IL, USA) according to Crofts et al.13) (link) Western blotting following native-PAGE was performed as described in Crofts et al.,13) (link) and Miura et al.16) (link)
Soluble protein (SP), loosely-bound protein (LBP) and tightly-bound protein (TBP) fractions were extracted from three developing endosperms as described by Fujita et al.20) (link) Western blotting was performed as described previously.21) (link)
A 700 mg aliquot of developing endosperms was extracted and fractionated by gel filtration chromatography using Superdex 200 resin packed in a XK16 column (16/400, GE Healthcare, Chicago, IL, USA) according to Crofts et al.13) (link) Western blotting following native-PAGE was performed as described in Crofts et al.,13) (link) and Miura et al.16) (link)
8
Formate Dehydrogenase Purification Protocol
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