Iodine-124 labeled A11 minibody ([124I]A11 Mb) was prepared by direct iodination using Pierce® Pre-coated Iodination Tubes (Thermo Scientific) according to the manufacturer’s instructions. Approximately 100 μg of protein were incubated with 15 MBq (400 μCi) of Na[124I]I (IBA Molecular) in 0.1 TRIS, pH 8.0 as previously described [10 (link)].
Pierce pre coated iodination tubes
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Pre-Coated Iodination Tubes are laboratory equipment designed for the iodination of proteins. The tubes are pre-coated with iodinating reagents, facilitating the iodination process and providing a convenient solution for researchers.
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Lab products found in correlation
Na125i radionuclide, by PerkinElmer (2 mentions)
Atomlab 400 dose calibrator, by Mirion Technologies (2 mentions)
Wallac wizard 1480, by PerkinElmer (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Wizard2, by PerkinElmer (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Ar 2000, by Bioscan (1 mentions)
Instant thin layer chromatography, by Agilent Technologies (1 mentions)
Cos 7 cells, by American Type Culture Collection (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (1 mentions)
Vasopressin, by Bio-Techne (1 mentions)
Ptre2 hygromycin vector, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Wallac wizard 1480 automatic gamma counter, by PerkinElmer (1 mentions)
Sodium metabisulfite, by Merck Group (1 mentions)
Chloramine t, by Thermo Fisher Scientific (1 mentions)
125i stock solution, by PerkinElmer (1 mentions)
Nap 5 size exclusion column, by Cytiva (1 mentions)
Chloramine t, by Merck Group (1 mentions)
Protein lobind tube, by Eppendorf (1 mentions)
13 protocols using pierce pre coated iodination tubes
1
Iodine-124 Labeled A11 Minibody Preparation
2
Radiolabeling and Pharmacokinetics of Fusion Proteins
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Tyrosine residues on GLP1-ELP, ELP-FGF21, or GLP1-ELP-FGF21 fusion proteins were reacted with Na125I radionuclide (PerkinElmer) using Pierce Pre-Coated Iodination Tubes (Thermo Fisher Scientific) and the indirect method for iodination. Radiolabeled protein was purified from unreacted radionuclide with Zeba Spin Desalting Columns (Thermo Fisher Scientific). Activities of radiolabeled constructs were measured with the Atomlab 400 Dose Calibrator (Biodex, Shirley, NY) and correlated to protein concentration. Mice received a single subcutaneous injection of radiolabeled fusion, and 10 μl of blood samples were collected at frequent time points from the tail vein and stored at room temperature until radioactivity quantification. Sample counts were measured at the end of the study on a Wallac Wizard 1480 automatic gamma counter (PerkinElmer). An activity-versus-count standard curve was used to convert sample counts to activities and subsequently to moles of drug.
3
Radioiodination and Pharmacokinetics of rhThrombin
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For iodination, lyophilized powder of rhThrombin was used. RhThrombin was iodinated with Na125I (Institute of Isotopes, Budapest, Hungary) IODO-GEN method according to Markwell and Fox (1978 (link)) using Pierce pre-coated iodination tubes (Thermo Scientific). The radioactivity of iodide-125 was measured using the automatic gamma counter Wizard 2 (PerkinElmer, USA).
The distribution experiments were conducted to determine systemic uptake of [125I]-rhThrombin from its formulation received in situ in Wistar male rats. In the hepatic wound model a cut was made on the lobe of the liver. The radiolabeled solution soaked gauze (1 cm2) at the dose of 1025 U and 315 µg proteins (∼10 µCi), the hydrogels (thermo-sensitive gel with 16% w/w poloxamer) and 1.5% w/w carbomer at the dose of 500 U and 155 µg proteins (∼5 µCi) were applied to the injury site for 5 min exposure; liver tissue content and the absorption from tissue to systemic circulation were measured up to 24 h (3 animals/time point). Specifically the goals of distribution experiments were to elucidate the pharmacokinetic parameters, which were calculated by the licensed software Kinetica™ TermoFisher.
The distribution experiments were conducted to determine systemic uptake of [125I]-rhThrombin from its formulation received in situ in Wistar male rats. In the hepatic wound model a cut was made on the lobe of the liver. The radiolabeled solution soaked gauze (1 cm2) at the dose of 1025 U and 315 µg proteins (∼10 µCi), the hydrogels (thermo-sensitive gel with 16% w/w poloxamer) and 1.5% w/w carbomer at the dose of 500 U and 155 µg proteins (∼5 µCi) were applied to the injury site for 5 min exposure; liver tissue content and the absorption from tissue to systemic circulation were measured up to 24 h (3 animals/time point). Specifically the goals of distribution experiments were to elucidate the pharmacokinetic parameters, which were calculated by the licensed software Kinetica™ TermoFisher.
4
Iodination and Characterization of LDL
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Human LDL was iodinated using Pierce pre-coated iodination tubes (Thermo Fisher) according to the manufacturer’s instructions. Each tube, in a final volume of 500 µl of solution A, contained human LDL (5 mg) and [125I]NaI (2 mCi). After incubation for 15 min at room temperature, 2 ml of solution A was added to the tube, and the entire mixture was loaded onto a PD-10 column (GE Healthcare) that had been pre-equilibrated with solution A. Elution fractions containing [125I]LDL were pooled and subjected to dialysis for 16 hr against 6 L of solution A to further eliminate unincorporated [125I]NaI. The dialyzed [125I]LDL had a specific activity of 70.6 cpm/ng and was stored at 4°C. The uptake and proteolytic degradation of [125I]LDL by cultured CHO-K1 cells was measured using previously described methods (Goldstein and Brown, 1974 (link)).
5
Validating JAM-A mAb/IR700 Binding Assay
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As a quality control of the JAM-A mAb/IR700, the levels of immune-binding of intact JAM-A mAb and JAM-A mAb/IR700 conjugates to alive PC3pip cells in vitro were assessed. First, JAM-A mAb and JAM-A mAb/IR700 conjugates were labeled with 125I Na using a direct protein iodination. Briefly, aliquots of 250-μCi 125I Na (PerkinElmer, Akron, OH) in Tris-Iodination Buffer were activated in the Pierce Pre-Coated Iodination Tubes (Thermo Scientific, Rockford, IL) followed by mixing with 40-μg of JAM-A mAbs or IR700 conjugates per tube. After incubation with Scavenging Buffer, mixes were purified with Zeba spin desalting columns at 1000 g for 2-min and elution fractions were collected. Next, for immune-binding assay, the purified radiolabeled JAM-A mAbs or IR700 conjugates were mixed with alive culture of PC3pip cells for 1-h at 4 °C followed by washing and radioactive scan of the collected pellets (Bioscan AR 2000, Bioscan Inc., Washington, DC). PC3pip cells that were blocked by an excess of JAM-A mAb prior these procedures were used as internal controls.
6
Radiolabeling of Trastuzumab with 131I
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Radiolabeling of trastuzumab with 131I was achieved using the Pierce Pre-coated Iodination Tubes (Thermo scientific, U.S.A.) and carried out in accordance with the protocol provided by Thermo scientific27 (link). Briefly, the pierce pre-coated iodination tube was wetted with 1 ml of Tris iodination buffer and decanted. 500 µCi of 131I was added to the Pierce pre-coated iodination tube and activated for 5 min at room temperature. Subsequently, 100 µg of trastuzumab was added to the tubes and the reaction mixture was incubated for 10 min at room temperature. Radiolabeling purity was determined by instant thin-layer chromatography (Agilent Technologies) using saline. Incorporation purity was always exceeded 95%.
7
Quantifying IL-3 receptor binding affinity
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Saturation binding assays were performed on transfected COS-7 cells to assess low-affinity binding or FDH cell lines expressing IL3R (βc + IL3Rα P248L) hexamers or (βc + IL3Rα WT) dodecamers to assess high-affinity binding using radioiodinated IL3 as previously described (90, 94 (link)). COS-7 cells were electroporated with pSG5:IL3Rα plasmids encoding WT or mutant IL3Rα. Cell-surface expression of receptor subunits was confirmed by flow cytometry. IL3 was radioiodinated with 125I (PerkinElmer) using Pierce Precoated Iodination tubes (Thermo Scientific; refs. 47, 80 (link)). Dissociation constants were calculated using the EBDA and LIGAND programs (KELL Radlig; ref. 95 (link)). Statistical significance of differences in KD values between cells expressing WT or IL3Rα (P) was determined using a two-tailed unpaired t test. To assess the stability of IL3Rα P248L compared with WT IL3Rα at the cell surface, all cell-surface proteins were biotinylated according to a commercially available kit (Pierce, #S44390) in FDH cells expressing P248L or WT IL3R at 0 to 60 minutes after IL3 stimulation, followed by streptavidin pulldown enrichment and immunoblotting of pulldowns for IL3Rα using anti-IL3Rα 9F5 antibody.
8
Characterizing IL-3 Receptor Binding Kinetics
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COS-7 cells (ATCC CRL-1651) were electroporated with pcDNA3.1:IL3Rα plasmids encoding wild-type or mutant IL3Rα, either alone or in the presence of the pSG5H:βc plasmid encoding human βc60 (link). Cell-surface expression of receptor subunits was confirmed by flow cytometry. Wild-type IL-3 and IL-3 K116W were radioiodinated with 125I (PerkinElmer) using Pierce pre-coated iodination tubes (Thermo Scientific). Saturation binding assays were performed on transfected COS cells, CTLL-2 cell lines, TF-1 cells or TF-1Hi cells using radioiodinated IL-360 (link). Dissociation constants were calculated using the EBDA and LIGAND programs63 (link) (KELL Radlig, Biosoft, UK). Representative data from IL-3-binding assays on TF-1 and TF-1Hi cells lines were also analysed by nonlinear regression using GraphPad Prism and yielded essentially identical dissociation constants.
9
Pharmacoperone Antagonist Assay Protocol
10
Radiolabeling and Pharmacokinetic Evaluation of ELP-FGF21
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