COS-7 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Antibodies targeting SR-BI's C-terminal region (residues 450–509) or extracellular domain (residues 230–380) and anti-microtubule-associated protein 1A/1B-light chain 3B (LC3B) were obtained from Novus Biologicals, Inc. (Littleton, CO). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Cell Signaling Technology (Danvers, MA). Anti-ubiquitin antibody was from Thermo Fisher Scientific (Waltham, MA). Peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were obtained from Amersham–GE Healthcare (Chicago, IL). Fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG was purchased from BD Biosciences (Franklin Lakes, NJ). Human HDL was purchased from Alfa Aesar (Tewksbury, MA). [3H]-cholesterol and [125I]-sodium iodide were from PerkinElmer (Waltham, MA). [3H]-cholesteryl oleyl ether (COE) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Chloroquine diphosphate salt, carbobenzoxy-Leu-Leu-leucinal (MG-132), cholesterol oxidase (Streptomyces), and thin-layer chromatography standards (cholesterol, 4-cholesten-3-one, and cholesteryl oleate) were obtained from Sigma–Aldrich (St. Louis, MO). All other reagents were of analytical grade.
125i sodium iodide
Manufactured by PerkinElmer
Sourced in United States
[125I]Sodium iodide is a radioactive isotope of iodine used as a tracer in various laboratory applications. It serves as a versatile tool for researchers, providing a means to label and track specific molecules or substances within experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
3h cholesterol, by PerkinElmer (5 mentions)
Cos 7 cells, by American Type Culture Collection (3 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by NOF corporation (3 mentions)
Cholesteryl oleate, by Merck Group (2 mentions)
Polyclonal anti sr bi, by Novus Biologicals (2 mentions)
3h cholesteryl oleoyl ether, by American Radiolabeled Chemicals (2 mentions)
Fugene 6 transfection reagent, by Promega (2 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (2 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Cell Signaling Technology (1 mentions)
Anti ubiquitin antibody, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated anti rabbit igg, by Cytiva (1 mentions)
Chloroquine diphosphate salt, by Merck Group (1 mentions)
3h cholesteryl oleyl ether, by American Radiolabeled Chemicals (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by GE Healthcare (1 mentions)
4 cholesten 3 one, by Merck Group (1 mentions)
Aminoguanidine hydrochloride, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Cytiva (1 mentions)
Rabbit anti gapdh 2118 antibody, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated donkey anti rabbit igg, by GE Healthcare (1 mentions)
Nb400 101, by Novus Biologicals (1 mentions)
11 protocols using 125i sodium iodide
1
COS-7 Cells: Antibody and Radioactive Assays
2
Cholesterol Homeostasis Regulation Analysis
3
Antibody-based Analysis of Apolipoprotein Modifications
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The following antibodies were used: monoclonal anti-apoA-I (Santa Cruz Biotechnology, Inc), monoclonal anti-acrolein (Abcam), polyclonal anti-apoA-II (Abcam), polyclonal anti-SR-BI specific for the C-terminal or the extracellular domain (Novus Biologicals, Inc., Littleton, CO); peroxidase-conjugated bovine anti-goat secondary IgG (Santa Cruz Biotechnology, Inc.), peroxidase-conjugated goat anti-rabbit IgG (Amersham- GE Healthcare), anti-mouse (Amersham- GE Healthcare). [3H]Cholesteryl oleoyl ether (COE) was purchased from American Radiolabeled Chemicals, Inc (St. Louis, MO). [125I]Sodium iodide and [3H]cholesterol were purchased from Perkin-Elmer. Acrolein was purchased from Ultra Scientific (North Kingstown, RI). Aminoguanidine hydrochloride was purchased from Acros Organics (Morris Plains, NJ). All other reagents were of analytical grade.
4
Quantifying SR-BI Expression in COS-7 Cells
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COS-7 cells were obtained from the American Type Culture Collection. Rabbit polyclonal antibody targeting SR-BI’s C-terminal region (NB400–101) was purchased from Novus Biologicals. Rabbit-anti-GAPDH (#2118) antibody was obtained from Cell Signaling Technology. Horseradish peroxidase (HRP)–conjugated donkey-anti-rabbit-IgG was purchased from GE Healthcare Life Sciences. Fluorescein isothiocyanate (FITC)–conjugated goat-anti-rabbit IgG secondary antibody (#554020) was from BD Biosciences. [125I]-sodium iodide, [3H]-cholesteryl hexadecyl ether, [3H]-cholesteryl oleyl ether, and [3H]-cholesterol were purchased from PerkinElmer. Acyl-CoA cholesterol acyltransferase inhibitor (Sandoz 58–035) was obtained from MilliporeSigma. Human HDL was obtained from Alfa Aesar. Recombinant cholesteryl ester transfer protein was from Roar Biomedical. Cholesterol oxidase from Streptomyces spp. and thin-layer chromatography standards (cholesterol, 4-cholesten-3-one, and cholesteryl oleate) were obtained from Sigma-Aldrich. FuGENE 6 transfection reagent was obtained from Promega. EZ-Link Sulfo-NHS-LC-Biotin was purchased from Thermo Fisher Scientific. All other reagents were of analytical grade.
5
Characterization of SR-BI Protein in COS-7 Cells
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COS-7 cells were obtained from ATCC (Manassas, VA). Rabbit polyclonal antibodies targeting the C-terminal region of SR-BI (amino acids 450–509; NB400-101) or near-C-terminal extracellular domain (amino acids 230–380; NB400-134) were purchased from Novus Biologicals (Littleton, CO). The anti-rabbit-GAPDH (#2118) antibody was obtained from Cell Signaling Technology (Danvers, MA). HRP-conjugated donkey-anti-rabbit-IgG secondary antibody was purchased from GE Healthcare Life Sciences (Marlborough, MA). [125I]sodium iodide, [3H]cholesteryl hexadecyl ether (CHE), and [3H]cholesterol were purchased from PerkinElmer (Waltham, MA). Human HDL and ACAT inhibitor (Sandoz 58-035) were obtained from MilliporeSigma (Burlington, MA). Recombinant cholesteryl ester transfer protein was from Roar Biomedical (New York, NY). Cholesterol oxidase from Streptomyces sp. and TLC standards (cholesterol, 4-cholesten-3-one, and cholesteryl oleate) were obtained from Sigma-Aldrich (St. Louis, MO). FuGENE 6 transfection reagent was obtained from Promega (Madison, WI). EZ-Link Sulfo-NHS-LC-Biotin and DiI-LDL were purchased from Thermo Scientific (Waltham, MA). DiI-HDL was obtained from Kalen Biomedical (Germantown, MD). All other reagents were of analytical grade.
6
Radiolabeled Liposome Formulation Protocol
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DOX was purchased from LC Laboratories (Woburn, MA, USA). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEG-DSPE) were purchased from NOF Corporation (Tokyo, Japan). Cholesterol (Chol) was purchased from Sigma Aldrich (St. Louis, MO, USA). [125I]Sodium iodide (644 GBq/mg) was purchased from PerkinElmer (Waltham, MA, USA). Other reagents were of reagent grade and used as received. Thin layer chromatography (TLC) was performed on silica plates 60 F254 (Merck, Darmstadt, Germany). Nuclear magnetic resonance (NMR) spectra were recorded on a JNM ECS400 (400 MHz) or JNM ECA600 (600 MHz) spectrometer (JEOL Ltd., Tokyo, Japan). The mass spectrometry was performed on a JMS T700 (JEOL Ltd.,) on electrospray ionization mass spectrometry (ESI-MS). HPLC analyses and purification were carried out on a Shimadzu Prominence HPLC system (Shimadzu Corp., Kyoto, Japan). Radioactivity was determined using an auto gamma counter ARC 7010B (Hitachi, Ltd., Tokyo, Japan). A colorectal adenocarcinoma cell line, Colon 26, was obtained from Cell Resource Centre for Biomedical Research in Tohoku University.
7
Recombinant β₂-Microglobulin Variants
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Recombinant wild type and D76N β2m were expressed in transformed E. coli BL21DE3 strains and purified to homogeneity by sequential gel filtration and anion exchange chromatography2 (link)20 (link).
Uniformly 15N- labeled wild type or D76N β2m isoforms were also prepared using Spectra 9 minimal medium for NMR analysis.
Both wild type and D76N β2m were labelled with 125I using N-bromosuccinimide and sodium [125I] iodide (Perkin Elmer, Seer Green, UK) in PBS for 10–15 s and purified on a PD10 desalting column (Bio-Rad, Hemel Hempstead, UK)21 (link); 125I-β2m was prepared at a specific activity of 16.0 MBq/mg. Approximately 1 μg (0.017 MBq) of 125I β2m was diluted into 100 μg of native recombinant β2m to give a final specific activity of ~0.17 MBq/mg for each mouse for in vivo clearance and tissue localization experiments.
The anti β2m nanobody, Nb24, originally selected from a phage display library constructed from a β2m immunized-camel, was expressed in E. coli as C-terminal His6-tagged protein using a pHEN6 cloning vector. The nanobody was purified to homogeneity by immobilized-metal affinity chromatography and gel filtration7 (link).
Uniformly 15N- labeled wild type or D76N β2m isoforms were also prepared using Spectra 9 minimal medium for NMR analysis.
Both wild type and D76N β2m were labelled with 125I using N-bromosuccinimide and sodium [125I] iodide (Perkin Elmer, Seer Green, UK) in PBS for 10–15 s and purified on a PD10 desalting column (Bio-Rad, Hemel Hempstead, UK)21 (link); 125I-β2m was prepared at a specific activity of 16.0 MBq/mg. Approximately 1 μg (0.017 MBq) of 125I β2m was diluted into 100 μg of native recombinant β2m to give a final specific activity of ~0.17 MBq/mg for each mouse for in vivo clearance and tissue localization experiments.
The anti β2m nanobody, Nb24, originally selected from a phage display library constructed from a β2m immunized-camel, was expressed in E. coli as C-terminal His6-tagged protein using a pHEN6 cloning vector. The nanobody was purified to homogeneity by immobilized-metal affinity chromatography and gel filtration7 (link).
8
Radiosynthesis of 125I-labeled ITdU
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Chemicals and solvents were purchased
from Sigma-Aldrich (St. Louis, MI, USA) and Merck (Darmstadt, Germany)
or otherwise as indicated. No-carrier-added (n.c.a.) sodium [125I]iodide was obtained from PerkinElmer (Waltham, MA, USA).
No-carrier-added Na125I was used as received. The 5-tributylstannyl
precursor of ITdU was synthesized according to previously reported
methods and provided by PD Dr. rer. nat. Boris Zlatopolskiy.15 (link) For labeling, 3 μL of a ITdU precursor
solution (123 mM in 66% MeOH aq) was added to 17 μL of buffer
(0.2 M phosphate buffer pH 2, 30% MeOH aq). Then, 10 μL of a
Na125I solution in 0.05 M NaOH was added before addition
of 16 μL Chloramine T solution (0.66 mg/mL in 66% methanol).
After 10 min at room temperature, the reaction mixture was purified
via high performance liquid chromatography (HPLC) on an analytical
C4 column (Multochrom-100-5 μ-C4, 250 mm × 4 mm, CS-Chromatographie,
Langerwehe, Germany). Quality control was performed by the same HPLC
method. After purification, the product fraction was concentrated
by dilution and sorption by a C18 cartridge (#WAT023501, Waters, Milford,
MA, USA), followed by rinsing, elution with 1 mL of MeCN, evaporation,
and dissolution in EtOH. Overall radiochemical yield (RCY) was >70%,
and final radiochemical purity was >95%. Mean product molar activities
were Am = 41 GBq/μmol.
from Sigma-Aldrich (St. Louis, MI, USA) and Merck (Darmstadt, Germany)
or otherwise as indicated. No-carrier-added (n.c.a.) sodium [125I]iodide was obtained from PerkinElmer (Waltham, MA, USA).
No-carrier-added Na125I was used as received. The 5-tributylstannyl
precursor of ITdU was synthesized according to previously reported
methods and provided by PD Dr. rer. nat. Boris Zlatopolskiy.15 (link) For labeling, 3 μL of a ITdU precursor
solution (123 mM in 66% MeOH aq) was added to 17 μL of buffer
(0.2 M phosphate buffer pH 2, 30% MeOH aq). Then, 10 μL of a
Na125I solution in 0.05 M NaOH was added before addition
of 16 μL Chloramine T solution (0.66 mg/mL in 66% methanol).
After 10 min at room temperature, the reaction mixture was purified
via high performance liquid chromatography (HPLC) on an analytical
C4 column (Multochrom-100-5 μ-C4, 250 mm × 4 mm, CS-Chromatographie,
Langerwehe, Germany). Quality control was performed by the same HPLC
method. After purification, the product fraction was concentrated
by dilution and sorption by a C18 cartridge (#WAT023501, Waters, Milford,
MA, USA), followed by rinsing, elution with 1 mL of MeCN, evaporation,
and dissolution in EtOH. Overall radiochemical yield (RCY) was >70%,
and final radiochemical purity was >95%. Mean product molar activities
were Am = 41 GBq/μmol.
9
Radiolabeling of Gold Nanoparticles
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All chemicals and solvents were of reagent grade and were used without further purification, unless stated otherwise, and were commercially acquired from Aldrich Chemical Co. Solvents for high-performance liquid chromatography (HPLC) were HPLC-grade. For the preparation of aqueous solutions and for rinsing of gold nanoparticles, Milli-Q (DI) water (ρ < 18MΩ) was used. The amino acids used in this work were acquired from Novabiochem. The AuNP-TDOTA nanoparticles were synthesized according to previously published methods [28 (link)]. 67GaCl3 was prepared from 67Ga-citrate (acquired from Mallinckrodt) following a protocol previously described [60 (link)]. Sodium [125I] iodide was obtained from Perkin Elmer, USA, as a non-carrier added solution in 0.1 M aqueous NaOH with radionuclidic purity >99% and specific activity of 643.8 GBq/mg. [177Lu]Cl3 as a carrier-added (specific activity >500 GBq/mg Lu) solution in 0.04 M HCl was kindly provided by the Radioisotope Centre POLATOM, National Centre for Nuclear Research in Otwock, Poland.
10
Radiolabeling of Organic Compounds
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