Sodium iodide [131I] (half-life = 8.04 days) was purchased from the China Institute of Atomic Energy (Beijing, China). Radioiodination of mouse anti-TLR5 mAb (100 μg/ml; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) and mouse isotype IgG (1 mg/ml; Biosynthesis Biotechnology Co., Ltd., Beijing, China) with 131I was performed according to the iodogen method, as previously described [14 (link)]. Mouse IgG served as a specific control antibody. Radioiodinated anti-TLR5 mAb and IgG were separated from free iodine using size-exclusion columns (PD-10 Sephadex G-25, GE-Healthcare, Diegem, Belgium), and the flowthrough was collected in sequential fractions. The radioactivity and concentration were measured using a gamma counter (Capintec Inc., Ramsey, NJ, USA).
Pd 10 sephadex g 25
Manufactured by GE Healthcare
Sourced in Belgium, United States
The PD-10 Sephadex G-25 is a size exclusion chromatography column used for desalting and buffer exchange of small molecules and proteins. It is designed to separate compounds based on their molecular size, allowing the removal of salts, buffers, or other small molecules from the sample.
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6 protocols using pd 10 sephadex g 25
1
Radioiodination of Anti-TLR5 Monoclonal Antibody
2
Radioactive Labeling and Stability of Anti-TLR5 mAb
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Briefly, 10 μg of anti‐TLR5 mAb/IgG was added to 100 μL of 0.05 M phosphate buffered (PB, pH 7.4), followed by the addition of 11.1 MBq (300 μCi) of Na125I, the mixture was incubated for 20 minutes at room temperature, and then the reaction was stopped by adding 150 μL of 0.05 M pH 7.4 PB and incubated for another 10 minutes at room temperature.24 The labelled compound was purified with PD‐10 Sephadex G‐25 (GE Healthcare), and the labelled rate was calculated. The mixture of 0.9% saline and methanol with a volume ratio of 1:2 was used as an unfolding agent. The stability of 125I‐anti‐TLR5 mAb and 125I‐IgG was determined in PBS and serum.
3
Radiolabeled Exosomes for Imaging
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HYNIC-TEx-Cy7/HYNIC-AEx-Cy7 (5 mg/mL, 1 mL) were incubated with 1 mL of tricine/EDDA solution (20 mg/mL tricine, 10 mg/mL EDDA at pH 6–7; Sigma/Aldrich, St. Louis, Mo, USA). The 99Mo/99mTc generator (Beijing Atom High Tech, Being, China) was used to obtain a 740 MBq 99mTcO4− solution, and 20 μL of SnCl2·2H2O (1 mg/mL in 0.1 N HCl; Sigma/Aldrich) was added. After a 30-min incubation, exosomes were purified using PD10 Sephadex G-25 (GE, USA). Radio thin-layer chromatography (radio-TLC) was used to determine the radiochemical purity with silica gel paper strips (Gelman Sciences, Germany) as a stationary phase and saline as a mobile phase. The stability of exosomes in FBS for 6 h at 37 °C was analyzed using radio-TLC. FT-IR was used to verify whether the DSPE-PEG2000 materials were inserted onto the exosomes.
4
Zinc Binding Assay for FrmR Variants
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Experiments were carried out as described previously (50 (link)). FrmR, FrmRE64H, FrmRC35A, or FrmRH60L was incubated (120 min) with an excess of ZnCl2 in 100 mm NaCl, 400 mm KCl, 10 mm HEPES, pH 7.0, and an aliquot (0.5 ml) was resolved by size exclusion chromatography (PD10 Sephadex G25, GE Healthcare) in the same buffer conditions. Fractions were analyzed for zinc by inductively coupled plasma MS and for protein by a Bradford assay as described (50 (link)). The control experiments with FrmR and FrmRE64H (Fig. 4 E) are new unpublished data sets and are presented here to demonstrate reproducibility and for comparative purposes.
5
Recombinant Protein Purification Protocol
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The cloned DNA plasmids were transformed into a BL21-Codon Plus (DE3)-RIPL strain (Novagen) of Escherichia coli for expression. For protein induction, 1 mM of IPTG (Duchefa Biochemie) was added. The cells were harvested and resuspended with native lysis buffer (10 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and then sonicated. Lysates were centrifuged, and the supernatants were mixed with Ni-NTA resin (Qiagen). The recombinant proteins were purified using native wash buffer (30 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and native elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The eluted recombinant proteins were desalted with PD-10 Sephadex G-25 (GE Healthcare) using 10% glycerol PBS. The proteins were then mixed with SP Sepharose Fast Flow (GE Healthcare) in binding buffer (50 mM NaH2PO4, 200 mM NaCl, pH 6.0). After washing with the binding buffer, bound proteins were eluted with elution buffer (50 mM NaH2PO4, 2 M NaCl, pH 6.0) and desalted using PD-10 Sephadex G-25. The endotoxin level in the purified recombinant protein was within the safe range, approximately 6 EU/mL. There were no symptoms of the endotoxin-induced immune response in the animal experiment using recombinant proteins, such as anaphylactic shock.
6
Analyzing Metal Binding Profiles
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FrmR, FrmRE64H, or Zur were incubated (60 min) with an excess of ZnCl2, CuCl (>95% Cu(I)) or EDTA (as stated) in 100 mm NaCl, 400 mm KCl, and 10 mm HEPES, pH 7.0, and an aliquot (0.5 ml) was resolved by size-exclusion chromatography (PD-10 Sephadex G25, GE Healthcare) in the same buffer conditions. Fractions (0.5 ml) were analyzed for metal by ICP-MS and protein by the Bradford assay using known concentrations of FrmR, FrmRE64H, or Zur as standards. Failure to recover all of the copper during experimentation with FrmR or FrmRE64H suggests (at least) some competition from and copper binding by the Sephadex matrix.
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